Process for preparing metabolized conditioned growth media

ABSTRACT

Compositions comprising metabolized conditioned growth medium and/or metabolized cell extract and methods of use are described. The metabolized conditioned growth medium and metabolized cell extract compositions may be formulated with an acceptable carrier into injectable or topical formulations, for example, as a cream, lotion or gel, and may be used in cosmeceutical or pharmaceutical applications. The metabolized conditioned growth medium and metabolized cell extract may also be further processed to concentrate or reduce one or more factors or components contained within the metabolized conditioned growth medium or metabolized cell extract. The growth medium may be conditioned by any eukaryotic cell. The metabolized conditioned growth medium and metabolized cell extract may be used to prevent or treat a condition, for example, a skin condition.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.13/294,599 filed Nov. 11, 2011, which claims the benefit of U.S.Provisional Application No. 61/413,166, filed Nov. 12, 2010, the entiredisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The disclosure relates to compositions comprising metabolizedconditioned growth medium and/or metabolized cell extract, and methodsfor preventing or treating a condition, for example a skin condition, byadministering the compositions to a subject.

BACKGROUND OF THE INVENTION

Culture medium compositions typically include essential amino acids,salts, vitamins, minerals, trace metals, sugars, lipids and nucleosides.Cell culture medium attempts to supply the components necessary to meetthe nutritional needs required to grow cells in a controlled, artificialand in vitro environment. Nutrient formulations, pH, and osmolarity varyin accordance with parameters such as cell type, cell density, and theculture system employed. Many cell culture medium formulations aredocumented in the literature and a number of media are commerciallyavailable. Once the culture medium is incubated with cells, it is knownto those skilled in the art as “spent” or “conditioned medium”.Conditioned medium contains many of the original components of themedium, as well as a variety of cellular metabolites and secretedproteins, including, for example, growth factors, inflammatory mediatorsand other extracellular proteins.

SUMMARY OF THE INVENTION

In one embodiment, the disclosure provides a composition comprisingmetabolized conditioned growth medium and an acceptable carrier. In someembodiments, the metabolized conditioned growth medium is conditionedgrowth medium metabolized by yeast cells. In some embodiments, theconditioned growth medium is prepared by culturing cells in a growthmedium sufficient to meet the nutritional needs required to grow thecells in vitro to form a conditioned growth medium. In otherembodiments, the composition is a topical or injectable composition. Insome embodiments, the composition is used to treat or prevent a skincondition. In other embodiments, the skin condition is a cosmeticdefect, a congenital defect, hair loss or an acquired defect. In furtherembodiments, the skin condition is fine lines and wrinkles; age spotsand dyspigmentation; decreased skin texture, tone and elasticity;roughness and photo damage; decreased ability of skin to regenerateitself; environmental damage; decreased smoothness and tightness ofskin; age spots; fine and coarse lines and wrinkles; fine and coarseperiocular wrinkles; nasolabial folds; facial fine and coarse lines;decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof. In one embodiment, the composition rejuvenates sundamaged and aging skin; improves the appearance of fine lines andwrinkles; promotes cell renewal; diminishes the appearance of age spotsand dyspigmentation; improves skin tone, texture and elasticity; reducesroughness and photo damage; prevents or reduces environmental damage;plumps the skin; brightens the skin; lightens the skin; strengthens theability of skin to regenerate itself; improves the appearance of agespots; brightens and lightens age spots; improves skin firmness,elasticity, resiliency; smoothes, tightens, or fills in fine lines onthe skin; reduces the appearance of dark circles under the eye; improveslip texture or condition; enhances natural lip color; increases lipvolume; promotes epithelialization of post-procedure skin; restores theskin's barrier or moisture balance; improves the appearance of agespots; improves the appearance of skin pigmentation, or a combinationthereof. In one embodiment, the compositions reduce the appearance offine lines and wrinkles; diminish the appearance of age spots anddyspigmentation; improve skin texture, tone and elasticity; reduceroughness and photo damage; strengthen the ability of skin to regenerateitself; prevent or reduce environmental damage; smooth and tightensskin; brighten and lighten age spots; reduce fine and coarse lines andwrinkles; improve appearance of fine and coarse periocular wrinkles;improve appearance of nasolabial folds; improve perioral wrinkles;improve facial fine and coarse lines; improve skin tone, radiance andevenness; improve skin firmness; reduce tactile roughness; improve skintexture, overall photodamage, overall hyperpigmentation; globalimprovement; reduction in appearance of dark spots and/or patches;improve appearance of skin brightness and youthful appearance; improveoverall condition of skin; improve the appearance of photoaged skin;improve appearance of instrincally and extrinsically aged skin; improveskin cellular turnover; improve skin barrier; improve skin's ability toretain moisture; reduce the appearance of brown and red blotchiness,redness; increase skin epidermal thickness; strengthen dermal epidermaljunction; reduce the appearance of pore size and pores; improvesmoothness, or a combination thereof.

In certain embodiments, administration of a composition described hereinmay result in at least a 2-fold improvement of one or more symptoms orconditions. Folds improvement of one or more symptoms or conditionsinclude, but are not limited to, 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 75-fold, 100-fold or more, or any number therebetween.In certain embodiments, administration of a composition described hereinmay result in improvement of about 1% to about 100%, about 10% to about90%, about 20% to about 80%, about 30% to about 70%, about 40% to about60%, or about 50%. In other embodiments, administration of a compositiondescribed herein may result in improvement of one or more symptoms orconditions of about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 100%, about 125%, about 150% or more of one or more symptoms orconditions.

In other embodiments, the disclosure also provides a compositioncomprising metabolized cell extract and an acceptable carrier. In someembodiments, the metabolized cell extract is cell extract metabolized byyeast cells. In some embodiments, the cell extract is derived fromanimal cells, skin cells or fibroblasts. In other embodiments, thecomposition is a topical or injectable composition. In some embodiments,the composition is used to treat or prevent a skin condition. In otherembodiments, the skin condition is a cosmetic defect, a congenitaldefect, hair loss or an acquired defect. In further embodiments, theskin condition is fine lines and wrinkles; age spots anddyspigmentation; decreased skin texture, tone and elasticity; roughnessand photo damage; decreased ability of skin to regenerate itself;environmental damage; decreased smoothness and tightness of skin; agespots; fine and coarse lines and wrinkles; fine and coarse periocularwrinkles; nasolabial folds; facial fine and coarse lines; decreased skinradiance and evenness; decreased skin firmness; hyperpigmentation; darkspots and/or patches; decreased skin brightness and youthful appearance;photoaged skin; intrinsically and extrinsically aged skin; abnormal skincellular turnover; decreased skin barrier; decrease of skin's ability toretain moisture; brown and red blotchiness; redness; abnormal skinepidermal thickness; reduction of dermal epidermal junction; increasedpore size and number of pores; or a combination thereof.

In one embodiment, the disclosure provides metabolized conditionedgrowth medium prepared by a process comprising: (a) culturing cells in agrowth medium sufficient to meet the nutritional needs required to growthe cells in vitro to form a conditioned growth medium and removing theconditioned growth medium from the cultured cells; (b) culturing yeastcells; (c) exposing the yeast cells to the conditioned growth medium;(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and (e) collecting the metabolizedconditioned growth medium.

In another embodiment, the disclosure provides processes for preparingmetabolized conditioned growth medium comprising: (a) culturing cells ina growth medium sufficient to meet the nutritional needs required togrow the cells in vitro to form a conditioned growth medium and removingthe conditioned growth medium from the cultured cells; (b) culturingyeast cells; (c) exposing the yeast cells to the conditioned growthmedium; (d) culturing the yeast cells to metabolize at least a portionof the conditioned growth medium; and (e) collecting the metabolizedconditioned growth medium.

In another embodiment, the disclosure provides compositions comprisingmetabolized conditioned growth medium, and an acceptable carrier. Inanother embodiment, the disclosure provides compositions comprisingmetabolized conditioned growth medium, and an acceptable carrier,wherein the topical composition is used to treat or prevent a skincondition. In some embodiments, the composition is used to treat orprevent a skin condition. In some embodiments, the skin condition is acosmetic defect, a congenital defect, hair loss or an acquired defect.In further embodiments, the skin condition is fine lines and wrinkles;age spots and dyspigmentation; decreased skin texture, tone andelasticity; roughness and photo damage; decreased ability of skin toregenerate itself; environmental damage; decreased smoothness andtightness of skin; age spots; fine and coarse lines and wrinkles; fineand coarse periocular wrinkles; nasolabial folds; facial fine and coarselines; decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof.

In another embodiment, the disclosure provides compositions comprisingmetabolized conditioned growth medium, and an acceptable carrier,wherein the topical composition is used to treat or prevent a skincondition, the metabolized conditioned growth medium is present in anamount of about 0.01% to about 50% by weight of the composition, and themetabolized conditioned growth medium is prepared by culturing yeastcells in a growth medium sufficient to meet the nutritional needsrequired to grow the cells in vitro to form a conditioned growth medium

In another embodiment, the disclosure provides methods for preventing ortreating a skin condition in a subject comprising administering to thesubject a therapeutically effective amount of metabolized conditionedgrowth medium. In some embodiments, the composition is used to treat orprevent a skin condition. In other embodiments, the skin condition is acosmetic defect, a congenital defect, hair loss or an acquired defect.In further embodiments, the skin condition is fine lines and wrinkles;age spots and dyspigmentation; decreased skin texture, tone andelasticity; roughness and photo damage; decreased ability of skin toregenerate itself; environmental damage; decreased smoothness andtightness of skin; age spots; fine and coarse lines and wrinkles; fineand coarse periocular wrinkles; nasolabial folds; facial fine and coarselines; decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof.

In one embodiment, the disclosure provides methods for treating hairloss comprising administering to a subject a therapeutically effectiveamount of metabolized conditioned growth medium. In another embodiment,the disclosure provides methods for stimulating hair growth comprisingadministering to a subject a therapeutically effective amount ofmetabolized conditioned growth medium. In another embodiment, thedisclosure provides methods for preventing or treating a congenitaldefect in a subject comprising administering to the subject atherapeutically effective amount of metabolized conditioned growthmedium. In another embodiment, the disclosure provides methods forpreventing or treating a cosmetic defect in a subject comprisingadministering to the subject a therapeutically effective amount ofmetabolized conditioned growth medium. In one embodiment, treatment ofthe cosmetic defect rejuvenates sun damaged and aging skin; improves theappearance of fine lines and wrinkles; promotes cell renewal; diminishesthe appearance of age spots and dyspigmentation; improves skin tone,texture and elasticity; reduces roughness and photo damage; prevents orreduces environmental damage; plumps the skin; brightens the skin;lightens the skin; strengthens the ability of skin to regenerate itself;improves the appearance of age spots; brightens and lightens age spots;improves skin firmness, elasticity, resiliency; smoothes, tightens, orfills in fine lines on the skin; reduces the appearance of dark circlesunder the eye; improves lip texture or condition; enhances natural lipcolor; increases lip volume; promotes epithelialization ofpost-procedure skin; restores the skin's barrier or moisture balance;improves the appearance of age spots; improves the appearance of skinpigmentation, or a combination thereof. In another embodiment, thedisclosure provides methods for preventing or treating an acquireddefect in a subject comprising administering to the subject atherapeutically effective amount of metabolized conditioned growthmedium. Said methods may rejuvenate sun damaged and aging skin; improvethe appearance of fine lines and wrinkles; promote cell renewal; improveskin tone, texture and/or firmness; plump the skin; brightens the skin;lighten the skin; strengthen the skin's ability to regenerate itself;improve the appearance of age spots; improve skin firmness, elasticity,resiliency; smooth, tighten, or fill in fine lines on the skin; reducethe appearance of dark circles under the eye; improve lip texture orcondition; enhance natural lip color; increases lip volume; promoteepithelialization of post-procedure skin; restore the skin's barrier ormoisture balance; improve the appearance of age spots; improve theappearance of skin pigmentation, reduce the appearance of fine lines andwrinkles; diminish the appearance of age spots and dyspigmentation;improve skin texture, tone and elasticity; reduce roughness and photodamage; strengthen the ability of skin to regenerate itself; prevent orreduce environmental damage; smooth and tightens skin; brighten andlighten age spots, reduce in fine and coarse lines and wrinkles, improveappearance of fine and coarse periocular wrinkles, improve appearance ofnasolabial folds, improve perioral wrinkles, improve facial fine andcoarse lines, improve skin tone, radiance and evenness, improve skinfirmness, reduce tactile roughness, improve skin texture, overallphotodamage, overall hyperpigmentation, global improvement, reduce inappearance of dark spots and/or patches, improve appearance of skinbrightness and youthful appearance, improve overall condition of skin,improve the appearance of photoaged skin, improve appearance ofinstrincally and extrinsically aged skin, improve skin cellularturnover, improve skin barrier, improve skin's ability to retainmoisture, reduce the appearance of brown and red blotchiness, redness,increase skin epidermal thickness, strengthen dermal epidermal junction,reduce the appearance of pore size and pores, improve smoothness, or acombination thereof.

In another embodiment, the disclosure provides methods for reducing theappearance of fine lines and wrinkles comprising administering to asubject a therapeutically effective amount of metabolized conditionedgrowth medium. In another embodiment, the disclosure provides methodsfor diminishing the appearance of age spots and dyspigmentationcomprising administering to a subject a therapeutically effective amountof metabolized conditioned growth medium. In another embodiment, thedisclosure provides methods for improving skin texture, tone andelasticity comprising administering to a subject a therapeuticallyeffective amount of metabolized conditioned growth medium. In anotherembodiment, the disclosure provides methods for reducing roughness andphoto damage comprising administering to a subject a therapeuticallyeffective amount of metabolized conditioned growth medium. In anotherembodiment, the disclosure provides methods for strengthening theability of skin to regenerate itself comprising administering to asubject a therapeutically effective amount of metabolized conditionedgrowth medium. In another embodiment, the disclosure provides methodsfor preventing or reducing environmental damage comprising administeringto a subject a therapeutically effective amount of metabolizedconditioned growth medium. In another embodiment, the disclosureprovides methods for smoothing and tightening skin comprisingadministering to a subject a therapeutically effective amount ofmetabolized conditioned growth medium. In another embodiment, thedisclosure provides methods for brightening and lightening age spotscomprising administering to a subject a therapeutically effective amountof metabolized conditioned growth medium.

In another embodiment, the disclosure provides metabolized cell extractprepared by a process comprising: (a) providing a cell extract; (b)culturing yeast cells; (c) exposing the yeast cells to the cell extract;(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and (e) collecting the metabolized conditioned cellextract.

In another embodiment, the disclosure provides processes for preparingthe metabolized cell extract comprising: (a) providing a cell extract;(b) culturing yeast cells; (c) exposing the yeast cells to the cellextract; (d) culturing the yeast cells to metabolize at least a portionof the cell extract; and (e) collecting the metabolized cell extract.

In another embodiment, the disclosure provides compositions comprisingmetabolized cell extract, and an acceptable carrier. In anotherembodiment, the disclosure provides compositions comprising metabolizedcell extract, and an acceptable carrier, wherein the topical compositionis used to treat or prevent a skin condition. In some embodiments, thecomposition is used to treat or prevent a skin condition. In otherembodiments, the skin condition is a cosmetic defect, a congenitaldefect, hair loss or an acquired defect. In further embodiments, theskin condition is fine lines and wrinkles; age spots anddyspigmentation; decreased skin texture, tone and elasticity; roughnessand photo damage; decreased ability of skin to regenerate itself;environmental damage; decreased smoothness and tightness of skin; agespots; fine and coarse lines and wrinkles; fine and coarse periocularwrinkles; nasolabial folds; facial fine and coarse lines; decreased skinradiance and evenness; decreased skin firmness; hyperpigmentation; darkspots and/or patches; decreased skin brightness and youthful appearance;photoaged skin; intrinsically and extrinsically aged skin; abnormal skincellular turnover; decreased skin barrier; decrease of skin's ability toretain moisture; brown and red blotchiness; redness; abnormal skinepidermal thickness; reduction of dermal epidermal junction; increasedpore size and number of pores; or a combination thereof.

In another embodiment, the disclosure provides compositions comprisingmetabolized cell extract, and an acceptable carrier, wherein the topicalcomposition is used to treat or prevent skin condition, the metabolizedcell extract is present in an amount of about 0.01% to about 50% byweight of the composition, and the metabolized cell extract is cellextract metabolized by yeast cells. In other embodiments, the skincondition is a cosmetic defect, a congenital defect, hair loss or anacquired defect. In further embodiments, the skin condition is finelines and wrinkles; age spots and dyspigmentation; decreased skintexture, tone and elasticity; roughness and photo damage; decreasedability of skin to regenerate itself; environmental damage; decreasedsmoothness and tightness of skin; age spots; fine and coarse lines andwrinkles; fine and coarse periocular wrinkles; nasolabial folds; facialfine and coarse lines; decreased skin radiance and evenness; decreasedskin firmness; hyperpigmentation; dark spots and/or patches; decreasedskin brightness and youthful appearance; photoaged skin; intrinsicallyand extrinsically aged skin; abnormal skin cellular turnover; decreasedskin barrier; decrease of skin's ability to retain moisture; brown andred blotchiness; redness; abnormal skin epidermal thickness; reductionof dermal epidermal junction; increased pore size and number of pores;or a combination thereof. In one embodiment, treatment of the cosmeticdefect rejuvenates sun damaged and aging skin; improves the appearanceof fine lines and wrinkles; promotes cell renewal; diminishes theappearance of age spots and dyspigmentation; improves skin tone, textureand elasticity; reduces roughness and photo damage; prevents or reducesenvironmental damage; plumps the skin; brightens the skin; lightens theskin; strengthens the ability of skin to regenerate itself; improves theappearance of age spots; brightens and lightens age spots; improves skinfirmness, elasticity, resiliency; smoothes, tightens, or fills in finelines on the skin; reduces the appearance of dark circles under the eye;improves lip texture or condition; enhances natural lip color; increaseslip volume; promotes epithelialization of post-procedure skin; restoresthe skin's barrier or moisture balance; improves the appearance of agespots; improves the appearance of skin pigmentation, or a combinationthereof.

In another embodiment, the disclosure provides methods for preventing ortreating a skin condition in a subject comprising administering to thesubject a therapeutically effective amount of metabolized cell extract.In another embodiment, the disclosure provides methods for treating hairloss comprising administering to a subject a therapeutically effectiveamount of metabolized cell extract. In another embodiment, thedisclosure provides methods for stimulating hair growth comprisingadministering to a subject a therapeutically effective amount ofmetabolized cell extract. In another embodiment, the disclosure providesmethods for preventing or treating a congenital defect in a subjectcomprising administering to the subject a therapeutically effectiveamount of metabolized cell extract. In another embodiment, thedisclosure provides methods for preventing or treating a cosmetic defectin a subject comprising administering to the subject a therapeuticallyeffective amount of metabolized cell extract. In one embodiment,treatment of the cosmetic defect rejuvenates sun damaged and aging skin;improves the appearance of fine lines and wrinkles; promotes cellrenewal; diminishes the appearance of age spots and dyspigmentation;improves skin tone, texture and elasticity; reduces roughness and photodamage; prevents or reduces environmental damage; plumps the skin;brightens the skin; lightens the skin; strengthens the ability of skinto regenerate itself; improves the appearance of age spots; brightensand lightens age spots; improves skin firmness, elasticity, resiliency;smoothes, tightens, or fills in fine lines on the skin; reduces theappearance of dark circles under the eye; improves lip texture orcondition; enhances natural lip color; increases lip volume; promotesepithelialization of post-procedure skin; restores the skin's barrier ormoisture balance; improves the appearance of age spots; improves theappearance of skin pigmentation, or a combination thereof. In anotherembodiment, the disclosure provides methods for preventing or treating aacquired defect in a subject comprising administering to the subject atherapeutically effective amount of metabolized cell extract. Saidmethods may rejuvenate sun damaged and aging skin; improve theappearance of fine lines and wrinkles; promote cell renewal; improveskin tone, texture and/or firmness; plump the skin; brightens the skin;lighten the skin; strengthen the skin's ability to regenerate itself;improve the appearance of age spots; improve skin firmness, elasticity,resiliency; smooth, tighten, or fill in fine lines on the skin; reducethe appearance of dark circles under the eye; improve lip texture orcondition; enhance natural lip color; increases lip volume; promoteepithelialization of post-procedure skin; restore the skin's barrier ormoisture balance; improve the appearance of age spots; improve theappearance of skin pigmentation, reduce the appearance of fine lines andwrinkles; diminish the appearance of age spots and dyspigmentation;improve skin texture, tone and elasticity; reduce roughness and photodamage; strengthen the ability of skin to regenerate itself; prevent orreduce environmental damage; smooth and tightens skin; brighten andlighten age spots, reduce in fine and coarse lines and wrinkles, improveappearance of fine and coarse periocular wrinkles, improve appearance ofnasolabial folds, improve perioral wrinkles, improve facial fine andcoarse lines, improve skin tone, radiance and evenness, improve skinfirmness, reduce tactile roughness, improve skin texture, overallphotodamage, overall hyperpigmentation, global improvement, reduce inappearance of dark spots and/or patches, improve appearance of skinbrightness and youthful appearance, improve overall condition of skin,improve the appearance of photoaged skin, improve appearance ofinstrincally and extrinsically aged skin, improve skin cellularturnover, improve skin barrier, improve skin's ability to retainmoisture, reduce the appearance of brown and red blotchiness, redness,increase skin epidermal thickness, strengthen dermal epidermal junction,reduce the appearance of pore size and pores, improve smoothness, or acombination thereof.

In another embodiment, the disclosure provides methods for reducing theappearance of fine lines and wrinkles comprising administering to asubject a therapeutically effective amount of metabolized cell extract.In another embodiment, the disclosure provides methods for diminishingthe appearance of age spots and dyspigmentation comprising administeringto a subject a therapeutically effective amount of metabolized cellextract. In another embodiment, the disclosure provides methods forimproving skin texture, tone and elasticity comprising administering toa subject a therapeutically effective amount of metabolized cellextract. In another embodiment, the disclosure provides methods forreducing roughness and photo damage comprising administering to asubject a therapeutically effective amount of metabolized cell extract.In another embodiment, the disclosure provides methods for strengtheningthe ability of skin to regenerate itself comprising administering to asubject a therapeutically effective amount of metabolized cell extract.In another embodiment, the disclosure provides methods for preventing orreducing environmental damage comprising administering to a subject atherapeutically effective amount of metabolized cell extract. In anotherembodiment, the disclosure provides methods for smoothing and tighteningskin comprising administering to a subject a therapeutically effectiveamount of metabolized cell extract. In another embodiment, thedisclosure provides methods for brightening and lightening age spotscomprising administering to a subject a therapeutically effective amountof metabolized cell extract.

In one embodiment, also provided are compositions comprising ametabolized conditioned growth medium and an acceptable carrier, whereinthe metabolized conditioned growth medium is conditioned growth mediummetabolized by yeast cells. In one embodiment, the conditioned growthmedium is prepared by culturing cells in a growth medium sufficient tomeet the nutritional needs required to grow the cells in vitro to form aconditioned growth medium. In another embodiment, the composition is aninjectable composition or a topical composition. In yet anotherembodiment, the topical composition is an ointment, a cream, a hydrogel,or a lotion. In still another embodiment, the metabolized conditiongrowth medium is encapsulated within an encapsulant. In one embodiment,the encapsulant is at least one of liposomes, niosomes, sub-micronemulsions, polymeric encapsulates, gels, creams and lotions. In someembodiments, the composition is used to treat or prevent a skincondition. In other embodiments, the composition is used to treat orprevent fine lines and wrinkles; age spots and dyspigmentation;decreased skin texture, tone and elasticity; roughness and photo damage;decreased ability of skin to regenerate itself; environmental damage;decreased smoothness and tightness of skin; age spots; fine and coarselines and wrinkles; fine and coarse periocular wrinkles; nasolabialfolds; facial fine and coarse lines; decreased skin radiance andevenness; decreased skin firmness; hyperpigmentation; dark spots and/orpatches; decreased skin brightness and youthful appearance; photoagedskin; intrinsically and extrinsically aged skin; abnormal skin cellularturnover; decreased skin barrier; decrease of skin's ability to retainmoisture; brown and red blotchiness; redness; abnormal skin epidermalthickness; reduction of dermal epidermal junction; increased pore sizeand number of pores; or a combination thereof. In still otherembodiments, the composition is used to treat or prevent a cosmeticdefect, a congenital defect, hair loss or an aquired defect. In someembodiments, the cosmetic defect is a glabellar frown line, deepnasolabial crease, circum-oral geographical wrinkle, sunken cheeks ormammary hypoplasia. In other embodiments, treatment of the cosmeticdefect rejuvenates sun damaged and aging skin; improves the appearanceof fine lines and wrinkles; promotes cell renewal; diminishes theappearance of age spots and dyspigmentation; improves skin tone, textureand elasticity; reduces roughness and photo damage; prevents or reducesenvironmental damage; plumps the skin; brightens the skin; lightens theskin; strengthens the ability of skin to regenerate itself improves theappearance of age spots; brightens and lightens age spots; improves skinfirmness, elasticity, resiliency; smoothes, tightens, or fills in finelines on the skin; reduces the appearance of dark circles under the eye;improves lip texture or condition; enhances natural lip color; increaseslip volume; promotes epithelialization of post-procedure skin; restoresthe skin's barrier or moisture balance; improves the appearance of agespots; improves the appearance of skin pigmentation, or a combinationthereof. In some embodiments, the acquired defect is a medical conditionthat occurs post-trauma, post-surgery or post-infection. In otherembodiments, the acquired defect is a post-medical procedure defect. Inyet other embodiments, the acquired defect is a depressed scar,subcutaneous atropy, a keratotic lesion, enophthalmos in an unucleatedeye, acne pitting of the face, linear scleroderma with subcutaneousatrophy, saddle-nose deformity, Romberg's disease or unilateral vocalcord paralysis.

Also provided are compositions comprising a metabolized cell extract andan acceptable carrier, wherein the metabolized cell extract is cellextract metabolized by yeast cells. In some embodiments, the cellextract is derived from animal cells. In other embodiments, the cellextract is derived from skin cells. In another embodiment, the cellextract is derived from fibroblasts. In some embodiments, thecomposition is an injectable composition or a topical composition. Insome embodiments, the topical composition is an ointment, a cream, ahydrogel, or a lotion. In other embodiments, the metabolized conditiongrowth medium is encapsulated within an encapsulant. In one embodiment,the encapsulant is at least one of liposomes, niosomes, sub-micronemulsions, polymeric encapsulates, gels, creams and lotions. In someembodiments, the composition is used to treat or prevent a skincondition. In other embodiments, the skin condition is a cosmeticdefect, a congential defect, hair loss or an acquired defect. In furtherembodiments, the skin condition is fine lines and wrinkles; age spotsand dyspigmentation; decreased skin texture, tone and elasticity;roughness and photo damage; decreased ability of skin to regenerateitself; environmental damage; decreased smoothness and tightness ofskin; age spots; fine and coarse lines and wrinkles; fine and coarseperiocular wrinkles; nasolabial folds; facial fine and coarse lines;decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof.

In yet other embodiments, the composition is used to treat or prevent acosmetic defect, a congenital defect, hair loss or an aquired defect. Insome embodiments, the cosmetic defect is a glabellar frown line, deepnasolabial crease, circum-oral geographical wrinkle, sunken cheeks ormammary hypoplasia. In some embodiennts, treatment of the cosmeticdefect rejuvenates sun damaged and aging skin; improves the appearanceof fine lines and wrinkles; promotes cell renewal; diminishes theappearance of age spots and dyspigmentation; improves skin tone, textureand elasticity; reduces roughness and photo damage; prevents or reducesenvironmental damage; plumps the skin; brightens the skin; lightens theskin; strengthens the ability of skin to regenerate itself; improves theappearance of age spots; brightens and lightens age spots; improves skinfirmness, elasticity, resiliency; smoothes, tightens, or fills in finelines on the skin; reduces the appearance of dark circles under the eye;improves lip texture or condition; enhances natural lip color; increaseslip volume; promotes epithelialization of post-procedure skin; restoresthe skin's barrier or moisture balance; improves the appearance of agespots; improves the appearance of skin pigmentation, or a combinationthereof. In some embodiments, the acquired defect is a medical conditionthat occurs post-trauma, post-surgery or post-infection. In otherembodiments, the acquired defect is a post-medical procedure defect. Inyet other embodiments, the acquired defect is a depressed scar,subcutaneous atropy, a keratotic lesion, enophthalmos in an unucleatedeye, acne pitting of the face, linear scleroderma with subcutaneousatrophy, saddle-nose deformity, Romberg's disease or unilateral vocalcord paralysis. In still other embodiments, the composition is used totreat or prevent fine lines and wrinkles; age spots and dyspigmentation;decreased skin texture, tone and elasticity; roughness and photo damage;decreased ability of skin to regenerate itself; environmental damage;decreased smoothness and tightness of skin; age spots; fine and coarselines and wrinkles; fine and coarse periocular wrinkles; nasolabialfolds; facial fine and coarse lines; decreased skin radiance andevenness; decreased skin firmness; hyperpigmentation; dark spots and/orpatches; decreased skin brightness and youthful appearance; photoagedskin; intrinsically and extrinsically aged skin; abnormal skin cellularturnover; decreased skin barrier; decrease of skin's ability to retainmoisture; brown and red blotchiness; redness; abnormal skin epidermalthickness; reduction of dermal epidermal junction; increased pore sizeand number of pores; or a combination thereof.

Also provided herein is a metabolized conditioned growth medium preparedby a process comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium. In someembodiments, the yeast used in the process above is of the Pichia genus.In other embodiments, the yeast is Pichia pastoris. In otherembodiments, the cells in step (a) are skin cells. In yet otherembodiments, the cells in step (a) are fibroblasts. In still otherembodiments, the cells in step (a) are animal cells. In still otherembodiments, the process to prepare the metabolized conditioned growthmedium further comprises (e) processing the metabolized conditionedgrowth medium, wherein processing is concentrating, filtering,purifying, or a combination thereof. In some embodiments, themetabolized conditioned growth medium includes a continuous flow systemor a bioreactor system in the culturing step of (a) or (b). In yet otherembodiments, the cells are suspended or floated in the growth medium.

Provided herein is a process for preparing the metabolized conditionedgrowth medium above, comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium. In oneembodiment, a composition is provided comprising the metabolizedconditioned growth medium and an acceptable carrier. In someemboidments, the metabolized conditioned growth medium is present in anamount of about 0.0001% to about 95% by weight of the composition. Inother embodiments, the metabolized conditioned growth medium is presentin an amount of about 0.01% to about 50% by weight of the composition.In some embodiments, the metabolized conditioned growth medium ispresent in an amount of about 0.01% to about 10% by weight of thecomposition. In some embodiments, the metabolized conditioned growthmedium is present in an amount of about 10% to about 20% by weight ofthe composition. In some embodiments, the metabolized conditioned growthmedium is present in an amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90% or 95% by weight of the composition. In one non-limitingexample, the metabolized conditioned growth medium is present in thecomposition in an amount of about 10%, about 15%, or about 20% by weightof the composition. In other embodiments, the composition furthercomprises at least one of water, surfactants, emulsifiers, conditioners,emollients, waxes, oils, polymers, thickeners, fixatives, colorants,nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,antifungals, antimicrobials, steroidal hormones, antidandruff agents,anti-acne components, sunscreens, and preservatives. In someembodiments, the composition is an injectable composition or a topicalcomposition. In one embodiment, the topical composition is an ointment,a cream, a hydrogel, or a lotion. In other embodiments, the metabolizedconditioned growth medium is encapsulated within an encapsulant. In someembodiments, the encapsulant is at least one of liposomes, niosomes,sub-micron emulsions, polymeric encapsulates, gels, creams, and lotions.

Also provided herein is a metabolized cell extract prepared by a processcomprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract. In some embodiments, ttheyeast is of the Pichia genus. In other embodiments, the yeast is Pichiapastoris. In yet other embodiments, the cell extract is derived fromskin cells. In still other embodiments, the cell extract is derived fromfibroblasts. In still other embodiments, the cell extract is derivedfrom animal cells. In yet other embodiments, the process furthercomprises (e) processing the metabolized cell extract, whereinprocessing is concentrating, filtering, purifying, or a combinationthereof. In one embodiment, the culturing comprises a continuous flowsystem or a bioreactor system.

In still other embodiments, a process for preparing the metabolized cellextract above comprises:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract.

Also provided herein are compositions comprising: an acceptable carrierand a metabolized conditioned growth medium prepared by a processcomprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium. In someembodiments, the yeast is of the Pichia genus. In other embodiments, theyeast is Pichia pastoris. In some embodiments, the cells in step (a) areskin cells. In other embodiments, the cells in step (a) are fibroblasts.In yet another embodiment, the cells in step (a) are animal cells. Insome embodiments, the process further comprises (e) processing themetabolized conditioned growth medium, wherein processing isconcentrating, filtering, purifying, or a combination thereof. In someembodiments, the culturing of step (a) or (b) comprises a continuousflow system or a bioreactor system. In yet other embodiments, the cellsare suspended or floated in the growth medium. In still otherembodiments, the metabolized conditioned growth medium is present in anamount of about 0.0001% to about 95% by weight of the composition. Instill other embodiments, the metabolized conditioned growth medium ispresent in an amount of about 0.01% to about 50% by weight of thecomposition. In yet other embodiments, the metabolized conditionedgrowth medium is present in an amount of about 0.01% to about 10% byweight of the composition. In some embodiments, the metabolizedconditioned growth medium is present in an amount of about 0.01%, 0.5%,1.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% by weight of the composition. In onenon-limiting example, the metabolized conditioned growth medium ispresent in the composition in an amount of about 10%, about 15%, orabout 20% by weight of the composition. In still other embodiments, thecomposition further comprises at least one of water, surfactants,emulsifiers, conditioners, emollients, waxes, oils, polymers,thickeners, fixatives, colorants, nutraceuticals, cosmeceuticals,therapeutics, pharmaceuticals, anifungals, antimicrobials, steroidalhormones, antidandruff agents, anti-acne components, sunscreens, andpreservatives. In still other embodiments, the composition is aninjectable composition or a topical composition. In yet otherembodiments, the topical composition is an ointment, a cream, ahydrogel, or a lotion. In some embodiments, the metabolized conditionedgrowth medium is encapsulated within an encapsulant. In one embodiment,the encapsulant is at least one of liposomes, niosomes, sub-micronemulsions, polymeric encapsulates, gels, creams, and lotions.

Also provided herein are compositions comprising: an acceptable carrierand a metabolized cell extract prepared by a process comprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract. In some embodiments, theyeast is of the Pichia genus. In other embodiments, the yeast is Pichiapastoris. In other embodiments, the cell extract is derived from skincells. In yet other embodiments, the cell extract is derived fromfibroblasts. In still other embodiments, the cell extract is derivedfrom animal cells. In one embodiment, the process further comprises (e)processing the metabolized cell extract, wherein processing isconcentrating, filtering, purifying, or a combination thereof. Inanother embodiment, the culturing comprises a continuous flow system ora bioreactor system. In still other embodiments, the metabolizedconditioned growth medium is present in an amount of about 0.0001% toabout 95% by weight of the composition. In yet another embodiment, themetabolized cell extract is present in an amount of about 0.01% to about50% by weight of the composition. In still other embodiments, themetabolized cell extract is present in an amount of about 0.01% to about10% by weight of the composition. In some embodiments, the metabolizedcell extract is present in an amount of about 10% to about 20% by weightof the composition. In some embodiments, the metabolized cell extract ispresent in an amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or95% by weight of the composition. In one non-limiting example, themetabolized cell extract is present in the composition in an amount ofabout 10%, about 15%, or about 20% by weight of the composition. In oneembodiment, the composition further comprises at least one of water,surfactants, emulsifiers, conditioners, emollients, waxes, oils,polymers, thickeners, fixatives, colorants, nutraceuticals,cosmeceuticals, therapeutics, pharmaceuticals, anifungals,antimicrobials, steroidal hormones, antidandruff agents, anti-acnecomponents, sunscreens, and preservatives. In still other embodiments,the composition is an injectable composition or a topical composition.In yet another embodiment, the topical composition is an ointment, acream, a hydrogel, or a lotion. In one embodiment, the metabolizedconditioned growth medium is encapsulated within an encapsulant. Inanother embodiment, the encapsulant is at least one of liposomes,niosomes, sub-micron emulsions, polymeric encapsulates, gels, creams,and lotions.

Also provided herein are methods for preventing or treating a skincondition in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising: anacceptable carrier and a metabolized conditioned growth medium preparedby a process comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium. In oneembodiment, the skin condition is a cosmetic defect. In anotherembodiment, the condition is hair loss. In another embodiment, thecondition is a congenital defect or an acquired defect. In oneembodiment, the acquired defect is a medical condition that occurspost-trauma, post-surgery or post-infection. In another embodiment, theacquired defect is a post-medical procedure defect. In yet anotherembodiment, the acquired defect is a depressed scar, subcutaneousatrophy, a keratotic lesion, enophthalmos in an unucleated eye, acnepitting of the face, linear scleroderma with subcutaneous atrophy,saddle-nose deformity, Romberg's disease or unilateral vocal cordparalysis. In one embodiment, the cosmetic defect is a glabellar frownline, deep nasolabial crease, circum-oral geographical wrinkle, sunkencheeks or mammary hypoplasia. In certain embodiments, the condition isfine lines and wrinkles; age spots and dyspigmentation; decreased skintexture, tone and elasticity; roughness and photo damage; decreasedability of skin to regenerate itself; environmental damage; decreasedsmoothness and tightness of skin; age spots; fine and coarse lines andwrinkles; fine and coarse periocular wrinkles; nasolabial folds; facialfine and coarse lines; decreased skin radiance and evenness; decreasedskin firmness; hyperpigmentation; dark spots and/or patches; decreasedskin brightness and youthful appearance; photoaged skin; intrinsicallyand extrinsically aged skin; abnormal skin cellular turnover; decreasedskin barrier; decrease of skin's ability to retain moisture; brown andred blotchiness; redness; abnormal skin epidermal thickness; reductionof dermal epidermal junction; increased pore size and number of pores;or a combination thereof. In certain embodiments, administration of acomposition described herein may result in at least a 2-fold improvementof one or more symptoms or conditions. Folds improvement of one or moresymptoms or conditions include, but are not limited to, 3-fold, 5-fold,10-fold, 15-fold, 20-fold, 25-fold, 75-fold, 100-fold or more, or anynumber therebetween. In certain embodiments, administration of acomposition described herein may result in improvement of about 1% toabout 100%, about 10% to about 90%, about 20% to about 80%, about 30% toabout 70%, about 40% to about 60%, or about 50%. In other embodiments,administration of a composition described herein may result inimprovement of one or more symptoms or conditions of about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 100%, about 125%, about 150%or more of one or more symptoms or conditions. In some embodiments, theyeast is of the Pichia genus. In some embodiments, the yeast is Pichiapastoris. In one embodiment, the cells in step (a) are skin cells. Inanother embodiment, the cells in step (a) are fibroblasts. In someembodiments, the cells in step (a) are animal cells. In otherembodiments, the process further comprises (e) processing themetabolized conditioned growth medium, wherein processing isconcentrating, filtering, purifying, or a combination thereof. In oneembodiment, the culturing of step (a) or (b) comprises a continuous flowsystem or a bioreactor system. In another embodiment, the cells aresuspended or floated in the growth medium. In still other embodiments,the metabolized conditioned growth medium is present in the compositionin an amount of about 0.0001% to about 95% by weight of the composition.In another embodiment, the metabolized conditioned growth medium ispresent in the composition in an amount of about 0.01% to about 50% byweight of the composition. In one embodiment, the metabolizedconditioned growth medium is present in the composition in an amount ofabout 0.01% to about 10% by weight of the composition. In someembodiments, the metabolized conditioned growth medium is present in anamount of about 10% to about 20% by weight of the composition. In someembodiments, the metabolized conditioned growth medium is present in anamount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% by weightof the composition. In one non-limiting example, the metabolizedconditioned growth medium is present in the composition in an amount ofabout 10%, about 15%, or about 20% by weight of the composition. In someembodiments, the composition further comprises at least one of water,surfactants, emulsifiers, conditioners, emollients, waxes, oils,polymers, thickeners, fixatives, colorants, nutraceuticals,cosmeceuticals, therapeutics, pharmaceuticals, anifungals,antimicrobials, steroidal hormones, antidandruff agents, anti-acnecomponents, sunscreens, and preservatives. In one embodiment, thecomposition is an injectable composition or a topical composition. Inanother embodiment, the topical composition is an ointment, a cream, ahydrogel, or a lotion. In one embodiment, the metabolized conditionedgrowth medium is encapsulated within an encapsulant. In someembodiments, the encapsulant is at least one of liposomes, niosomes,sub-micron emulsions, polymeric encapsulates, gels, creams, and lotions.In some embodiments, at least one extracellular matrix protein isup-regulated by the administration of the composition to the subject. Insome embodiments, the extracellular matrix protein is up-regulated byabout 5% to about 100%. In one embodiment, the extracellular matrixprotein is up-regulated by about 10% to about 50%. In anotherembodiment, the extracellular matrix protein is up-regulated by about60% to about 100%. In yet another embodiment, the extracellular matrixprotein is up-regulated by about 5%, by about 10%, by about 15%, byabout 20%, by about 25%, by about 30%, by about 35%, by about 40%, byabout 45%, by about 50%, by about 55%, by about 60%, by about 65%, byabout 70%, by about 75%, by about 80%, by about 85%, by about 90%, byabout 95% or by about 100%. In one embodiment, the extracellular matrixprotein is a collagen protein or a lysyl hydroxylase protein. In someembodiments, the extracellular matrix protein is encoded by COL4A1 geneor PLOD1 gene. In other embodiments, at least one repair protein isup-regulated by the administration of the composition to the subject. Inone embodiment, the repair protein is up-regulated by about 10% to about70%. In another embodiment, the repair protein is up-regulated by about25% to about 50%. In some embodiments, the repair-protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95% or byabout 100%. In one embodiment, the repair protein is a fibronectinprotein. In yet another embodiment, the repair protein is encoded by FN1gene. In some embodiments, at least one cellular connectivity protein isup-regulated by the administration of the composition to the subject. Inone embodiment, the cellular connectivity protein is up-regulated byabout 5% to about 200%. In one embodiment, the cellular connectivityprotein is up-regulated by about 10% to about 80%. In some embodiments,the cellular connectivity protein is up-regulated by about 30% to about50%. In one embodiment, the cellular connectivity protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95%, byabout 100%, by about 110%, by about 120%, by about 130%, by about 140%,by about 150%, by about 160%, by about 170%, by about 180%, by about190% or by about 200%. In other embodiments, the cellular connectivityprotein is involucrin protein. In some embodiments, the cellularconnectivity protein is encoded by IVL gene. In one embodiment, at leastone antioxidant protein is up-regulated by the administration of thecomposition to the subject. In yet another embodiment, the antioxidantprotein is up-regulated by about 5% to about 300%. In some embodiments,the antioxidant protein is up-regulated by about 10% to about 50%. Inother embodiments, the antioxidant protein is up-regulated by about 200%to about 250%. In some embodiments, the antioxidant protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95%, byabout 100%, by about 125%, by about 150%, by about 175%, by about 200%,by about 250% or by about 300%. In one embodiment, the antioxidantprotein is a superoxide dismutase protein. In another embodiment, theantioxidant protein is encoded by SOD2 gene.

Also provided herein are methods for preventing or treating a skincondition in a subject comprising administering to the subject atherapeutically effective amount of a composition comprising: anacceptable carrier and a metabolized cell extract prepared by a processcomprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract.

In one embodiment, the condition is a cosmetic defect. In anotherembodiment, the condition is hair loss. In another embodiment, thecondition is a congenital defect or an acquired defect. In oneembodiment, the acquired defect is a medical condition that occurspost-trauma, post-surgery or post-infection. In another embodiment, theacquired defect is a post-medical procedure defect. In yet anotherembodiment, the acquired defect is a depressed scar, subcutaneousatrophy, a keratotic lesion, enophthalmos in an unucleated eye, acnepitting of the face, linear scleroderma with subcutaneous atrophy,saddle-nose deformity, Romberg's disease or unilateral vocal cordparalysis. In one embodiment, the cosmetic defect is a glabellar frownline, deep nasolabial crease, circum-oral geographical wrinkle, sunkencheeks or mammary hypoplasia. In certain embodiments, the skin conditionis a cosmetic defect, a congenital defect, hair loss or an acquiredmedical defect. In further embodiments, the skin condition is fine linesand wrinkles; age spots and dyspigmentation; decreased skin texture,tone and elasticity; roughness and photo damage; decreased ability ofskin to regenerate itself; environmental damage; decreased smoothnessand tightness of skin; age spots; fine and coarse lines and wrinkles;fine and coarse periocular wrinkles; nasolabial folds; facial fine andcoarse lines; decreased skin radiance and evenness; decreased skinfirmness; hyperpigmentation; dark spots and/or patches; decreased skinbrightness and youthful appearance; photoaged skin; intrinsically andextrinsically aged skin; abnormal skin cellular turnover; decreased skinbarrier; decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof. In certain embodiments, administration of acomposition described herein may result in at least a 2-fold improvementof one or more symptoms or conditions. Folds improvement of one or moresymptoms or conditions include, but are not limited to, 3-fold, 5-fold,10-fold, 15-fold, 20-fold, 25-fold, 75-fold, 100-fold or more, or anynumber therebetween. In certain embodiments, administration of acomposition described herein may result in improvement of about 1% toabout 100%, about 10% to about 90%, about 20% to about 80%, about 30% toabout 70%, about 40% to about 60%, or about 50%. In other embodiments,administration of a composition described herein may result inimprovement of one or more symptoms or conditions of about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 100%, about 125%, about 150%or more of one or more symptoms or conditions. In some embodiments, theyeast is of the Pichia genus. In some embodiments, the yeast is Pichiapastoris. In one embodiment, the cells in step (a) are derived from skincells. In another embodiment, the cells in step (a) are derived fromfibroblasts. In some embodiments, the cells in step (a) are derived fromanimal cells. In other embodiments, the process further comprises (e)processing the metabolized cell extract, wherein processing isconcentrating, filtering, purifying, or a combination thereof. In oneembodiment, the culturing of step (a) or (b) comprises a continuous flowsystem or a bioreactor system. In another embodiment, the cells aresuspended or floated in the growth medium. In still other embodiments,the metabolized cell extract is present in the composition in an amountof about 0.0001% to about 95% by weight of the composition. In anotherembodiment, the metabolized cell extract is present in the compositionin an amount of about 0.01% to about 50% by weight of the composition.In one embodiment, the metabolized cell extract is present in thecomposition in an amount of about 0.01% to about 10% by weight of thecomposition. In some embodiments, the metabolized cell extract ispresent in an amount of about 10% to about 20% by weight of thecomposition. In some embodiments, the metabolized cell extract ispresent in an amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or95% by weight of the composition. In one non-limiting example, themetabolized cell extract is present in the composition in an amount ofabout 10%, about 15%, or about 20% by weight of the composition. In someembodiments, the composition further comprises at least one of water,surfactants, emulsifiers, conditioners, emollients, waxes, oils,polymers, thickeners, fixatives, colorants, nutraceuticals,cosmeceuticals, therapeutics, pharmaceuticals, anifungals,antimicrobials, steroidal hormones, antidandruff agents, anti-acnecomponents, sunscreens, and preservatives. In one embodiment, thecomposition is an injectable composition or a topical composition. Inanother embodiment, the topical composition is an ointment, a cream, ahydrogel, or a lotion. In one embodiment, the metabolized cell extractis encapsulated within an encapsulant. In some embodiments, theencapsulant is at least one of liposomes, niosomes, sub-micronemulsions, polymeric encapsulates, gels, creams, and lotions. In someembodiments, at least one extracellular matrix protein is up-regulatedby the administration of the composition to the subject. In someembodiments, the extracellular matrix protein is up-regulated by about5% to about 100%. In one embodiment, the extracellular matrix protein isup-regulated by about 10% to about 50%. In another embodiment, theextracellular matrix protein is up-regulated by about 60% to about 100%.In yet another embodiment, the extracellular matrix protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95% or byabout 100%. In one embodiment, the extracellular matrix protein is acollagen protein or a lysyl hydroxylase protein. In some embodiments,the extracellular matrix protein is encoded by COL4A1 gene or PLOD1gene. In other embodiments, at least one repair protein is up-regulatedby the administration of the composition to the subject. In oneembodiment, the repair protein is up-regulated by about 10% to about70%. In another embodiment, the repair protein is up-regulated by about25% to about 50%. In some embodiments, the repair-protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95% or byabout 100%. In one embodiment, the repair protein is a fibronectinprotein. In yet another embodiment, the repair protein is encoded by FN1gene. In some embodiments, at least one cellular connectivity protein isup-regulated by the administration of the composition to the subject. Inone embodiment, the cellular connectivity protein is up-regulated byabout 5% to about 200%. In one embodiment, the cellular connectivityprotein is up-regulated by about 10% to about 80%. In some embodiments,the cellular connectivity protein is up-regulated by about 30% to about50%. In one embodiment, the cellular connectivity protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95%, byabout 100%, by about 110%, by about 120%, by about 130%, by about 140%,by about 150%, by about 160%, by about 170%, by about 180%, by about190% or by about 200%. In other embodiments, the cellular connectivityprotein is involucrin protein. In some embodiments, the cellularconnectivity protein is encoded by IVL gene. In one embodiment, at leastone antioxidant protein is up-regulated by the administration of thecomposition to the subject. In yet another embodiment, the antioxidantprotein is up-regulated by about 5% to about 300%. In some embodiments,the antioxidant protein is up-regulated by about 10% to about 50%. Inother embodiments, the antioxidant protein is up-regulated by about 200%to about 250%. In some embodiments, the antioxidant protein isup-regulated by about 5%, by about 10%, by about 15%, by about 20%, byabout 25%, by about 30%, by about 35%, by about 40%, by about 45%, byabout 50%, by about 55%, by about 60%, by about 65%, by about 70%, byabout 75%, by about 80%, by about 85%, by about 90%, by about 95%, byabout 100%, by about 125%, by about 150%, by about 175%, by about 200%,by about 250% or by about 300%. In one embodiment, the antioxidantprotein is a superoxide dismutase protein. In another embodiment, theantioxidant protein is encoded by SOD2 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph representing the kinetics of the deposition ofglycosaminoglycans and collagen laid down over time by thethree-dimensional tissue products Transcyte™ and Dermagraft®. Thedeposition volumes of the glycosaminoglycans are dependent on the periodof growth while the deposition of collagen is not dependent on theperiod of growth.

FIG. 2 is a graph representing the effect of extracellular matrix(removed from Transcyte™) and added at dilutions of 1:2, 1:5, 1:10, and1:100 to monolayer cultures of human fibroblasts and keratinocytes. Themost significant effect illustrated is at a 1:10 dilution of the matrix.

FIG. 3 is a graph representing relative proliferation of humanfibroblasts and keratinocytes exposed to conditioned medium (cellculture medium which has previously supported the growth of cells inTranscyte™). An increase in cell response was revealed in as little asthree days.

FIG. 4 is a graph demonstrating investigator assessments of fine andcoarse wrinkles Significant reductions in mean scores were observed forfine and coarse wrinkles at all visits (all P<0.0001). In each data set,fine wrinkle assessment is shown on the left and coarse wrinkleassessment is shown in the right.

FIG. 5 a graph demonstrating investigator assessments of skin tone,firmness and radiance. Significant improvements in mean scores for skinfirmness, skin tone and radiance at all visits (all P≦0.02). In eachdata set, week 4 results are shown on the left, week 8 results are shownin the middle and week 12 results are shown on the right.

FIG. 6 is a graph demonstrating investigator assessments of tactileroughness. Significant improvements in mean scores for skin firmness,skin tone and radiance at all visits (all P<0.0001). Percent change frombaseline at week 4 is shown in the left-hand column, week 8 in themiddle column and week 12 in the right-hand column, respectively.

FIG. 7 is a graph providing the results at week 12 of a subjectself-assessment questionnaire from the 12-week clinical usage(Combined/Combination Product) study described in Example 18.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to novel compositions comprisingmetabolized conditioned growth medium and/or metabolized cell extract,cultured using any eukaryotic cell type or three-dimensional tissueconstruct, and methods for using the compositions. The cells arecultured in monolayer or in three-dimensions. The cells are preferablyhuman and include stromal cells, parenchymal cells, mesenchymal stemcells, liver reserve cells, neural stem cells, pancreatic stem cellsand/or embryonic stem cells. Medium conditioned by cell and tissuecultures will contain a variety of naturally secreted proteins, such asbiologically active growth factors. Also disclosed are novelcompositions comprising products derived from the conditioned cell mediaand uses for these compositions.

The “pre-conditioned” cell culture medium may be any cell culture mediumwhich adequately addresses the nutritional needs of the cells beingcultured. Examples of cell media include, but are not limited toDulbecco's Modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640,Iscove's, McCoy's and other media formulations readily apparent to thoseskilled in the art, including those found in Methods For Preparation ofMedia, Supplements and Substrate For Serum-Free Animal Cell Culture AlanR. Liss, New York (1984) and Cell & Tissue Culture: LaboratoryProcedures, John Wiley & Sons Ltd., Chichester, England 1996, both ofwhich are incorporated by reference herein in their entirety. The mediummay be supplemented, with any components necessary to support thedesired cell or tissue culture. Additionally serum, such as bovineserum, which is a complex solution of albumins, globulins, growthpromoters and growth inhibitors may be added if desired. The serumshould be pathogen free and carefully screened for mycoplasma bacterial,fungal, and viral contamination. Also, the serum should generally beobtained from the United States and not obtained from countries whereindigenous livestock carry transmittable agents. Hormone addition intothe medium may or may not be desired. The medium may also be serum-free,i.e., cells are grown in the absence of a serum supplement.

The “conditioned growth medium” may be any growth medium conditioned asdisclosed herein, and can be prepared, for example, as disclosed in U.S.Pat. Nos. 6,372,494; 7,118,746; 7,160,726, all of which are incorporatedherein by reference in its entirety.

Other ingredients, such as vitamins, growth and attachment factors,proteins etc., can be selected by those of skill in the art inaccordance with his or her particular need. In some embodiments, anycell type appropriate to achieve the desired conditioned medium may beused. Genetically engineered cells may be used to culture the media.Such cells can be modified, for example, to express a desired protein orproteins so that the concentration of the expressed protein or proteinsin the medium is optimized for the particular desired application. Inaccordance with the present disclosure, the cells and tissue culturesused to condition the medium may be engineered to express a target geneproduct which may impart a wide variety of functions, including but notlimited to, improved properties in expressing proteins resemblingphysiological reactions, increased expression of a particular proteinuseful for a specific application, such as wound healing or inhibitingcertain proteins such as proteases, lactic acid, etc.

The cells may be engineered to express a target gene product which isbiologically active which provides a chosen biological function, whichacts as a reporter of a chosen physiological condition, which augmentsdeficient or defective expression of a gene product, or which providesan anti-viral, anti-bacterial, anti-microbial, or anti-cancer activity.In accordance with the present disclosure, the target gene product maybe a peptide or protein, such as an enzyme, hormone, cytokine, antigen,or antibody, a regulatory protein, such as a transcription factor or DNAbinding protein, a structural protein, such as a cell surface protein,or the target gene product may be a nucleic acid such as a ribosome orantisense molecule. The target gene products include, but are notlimited to, gene products which enhance cell growth. For example, thegenetic modification may upregulate an endogenous protein, introduce anew protein or regulate ion concentration by expressing a heterologousion channel or altering endogenous ion channel function. Examplesinclude, but are not limited to engineered tissues that express geneproducts which are delivered systemically (e.g., secreted gene productssuch as proteins including growth factors, hormones, Factor VIII, FactorIX, neurotransmitters, and enkaphalins).

In some embodiments, cells are grown in a two-dimensional monolayeraccording to any method known to the skilled artisan. See, e.g., cellculture techniques such as those described in Sambrook J et al. (2000)Molecular Cloning: A Laboratory Manual (Third Edition); Goeddel, ed.(1990) Methods in Enzymology 185, Current Protocols In MolecularBiology; F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.(1994, supplemented through 1999); Animal Cell Culture: A PracticalApproach (Freshney ed. 1986).

In some embodiments, cells are grown on a three-dimensional stromalsupport and grow in multiple layers, forming a cellular matrix. Thismatrix system approaches physiologic conditions found in vivo to agreater degree than previously described monolayer tissue culturesystems. Three-dimensional cultures, such as Dermagraft® (AdvancedTissue Sciences, Inc., La Jolla, Calif.)“Dermagraft®”, and TransCyte™(Smith & Nephew, PLC, United Kingdom) “Transcyte™”, produce numerousgrowth factors and other proteins that are secreted into the medium atphysiological ratios and concentrations. Dermagraft® is composed ofallogeneic neonatal fibroblasts cultured on biodegradable polyglactin.TransCyte™ is a temporary living skin replacement comprising athree-dimensional stromal tissue bonded to a transitional covering asdescribed in U.S. Pat. No. 5,460,939. Additionally, thethree-dimensional tissue cultures which condition the cell media maycontain mesenchymal stem cells, liver reserve cells, neural stem cells,pancreatic stem cells, and/or embryonic stem cells and/or parenchymalcells and/or parenchymal stem cells found in many tissue types,including but not limited to bone marrow, skin, liver, pancreas, kidney,adrenal and neurologic tissue, as well as tissues of thegastrointestinal and genitourinary tracts, and the circulatory system.See U.S. Pat. Nos. 4,721,096; 4,963,489; 5,032,508; 5,266,480;5,160,490; and 5,559,022, each of which is incorporated by referenceherein in their entirety.

I. DEFINITIONS

The following terms used herein shall have the meanings indicated:

Cells: includes cells from any organism, including animals, plants,fungi, protists, or monera. In one embodiment, the source of the cellsis an animal or a plant. In another embodiment, the animal is a mammal.In another embodiment, the mammal is a human.

Cell Extract: includes lysed or fragmented cell content that may or maynot include cell particulate matter.

Adherent Layer: cells attached directly to the three-dimensional supportor connected indirectly by attachment to cells that are themselvesattached directly to the support.

Conditioned Medium: a formulation containing extracellular protein(s)and cellular metabolites, which has previously supported the growth ofany desired eukaryotic cell type, said cells having been cultured ineither two or three dimensions. Also called “Conditioned Cell Medium” or“Conditioned Cell and Tissue Culture Medium”.

Stromal Cells: fibroblasts with or without other cells and/or elementsfound in loose connective tissue, including but not limited to,endothelial cells, pericytes, macrophages, monocytes, plasma cells, mastcells, adipocytes, mesenchymal stem cells, liver reserve cells, neuralstem cells, pancreatic stem cells, chondrocytes, prechondrocytes, etc.

Tissue-Specific or Parenchymal Cells: the cells which form the essentialand distinctive tissue of an organ as distinguished from its supportiveframework.

Two-Dimensional Cell Culture: cells cultured in a monolayer, forexample, on a flat surface or the like.

Three-Dimensional Framework: a three-dimensional scaffold composed ofany material and/or shape that (a) allows cells to attach to it (or canbe modified to allow cells to attach to it); and (b) allows cells togrow in more than one layer. This support is inoculated with stromalcells to form the living three-dimensional stromal tissue. The structureof the framework can include a mesh, a sponge or can be formed from ahydrogel.

Three-Dimensional Stromal Tissue or Living Stromal Matrix: athree-dimensional framework which has been inoculated with stromal cellsthat are grown on the support. The extracellular matrix proteinselaborated by the stromal cells are deposited onto the framework, thusforming a living stromal tissue. The living stromal tissue can supportthe growth of tissue-specific cells later inoculated to form thethree-dimensional cell culture.

Tissue-Specific Three-Dimensional Cell Culture or Tissue-SpecificThree-Dimensional Construct: a three-dimensional living stromal tissuewhich has been inoculated with tissue-specific cells and cultured. Ingeneral, the tissue specific cells used to inoculate thethree-dimensional stromal matrix should include the “stem” cells (or“reserve” cells) for that tissue; i.e., those cells which generate newcells that will mature into the specialized cells that form theparenchyma of the tissue.

The following abbreviations shall have the meanings indicated:

BCS=bovine calf serum

BFU-E=burst-forming unit-erythroid

TGF-β=transforming growth factor-β

CFU-C=colony forming unit-culture

CFU-GEMM=colony forming unit-granuloid, erythroid, monocyte,megakaryocyte

CSF=colony-stimulating factor

DMEM=Dulbecco's Modified Eagle's Medium

EDTA=ethylene diamine tetraacetic acid

FBS=fetal bovine serum

FGF=fibroblast growth factor

GAG=glycosaminoglycan

GM-C SF=granulocyte/macrophage colony-stimulating factor

HBSS=Hank's balanced salt solution

HS=horse serum

IGF=insulin-like growth factor

LTBMC=long term bone marrow culture

MEM=minimal essential medium

PBL=peripheral blood leukocytes

PBS=phosphate buffered saline

PDGF=platelet-derived growth factor

RPMI 1640=Roswell Park Memorial Institute medium number 1640 (GIBCO,Inc., Grand Island, N.Y.)

SEM=scanning electron microscopy

VEGF=vascular endothelial growth factor

II. METABOLIZED CONDITIONED GROWTH MEDIUM AND METABOLIZED CELL EXTRACT

In one embodiment, the disclosure provides metabolized conditionedgrowth medium prepared by a process comprising: (a) culturing cells in agrowth medium sufficient to meet the nutritional needs required to growthe cells in vitro to form a conditioned growth medium and removing theconditioned growth medium from the cultured cells; (b) culturing yeastcells; (c) exposing the yeast cells to the conditioned growth medium;(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and (e) collecting the metabolizedconditioned growth medium. In some embodiments, the cells may originatefrom a plant and/or animal source, including human.

In another embodiment, the disclosure provides metabolized cell extractprepared by a process comprising: (a) providing a cell extract; (b)culturing yeast cells; (c) exposing the yeast cells to the cell extract;(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and (e) collecting the metabolized cell extract. In someembodiments, the cell extract is obtained by: culturing cells in agrowth medium sufficient to meet the nutritional needs required to growthe cells in vitro and removing the resulting cell extract from thegrowth medium. In some embodiments, the cells may originate from a plantand/or animal source, including human.

Metabolizing the extracellular proteins in conditioned growth media orcell extracts, such as growth factors, cytokines, and stress proteins,opens new possibilities in the preparation of products for use in alarge variety of areas including tissue repair, e.g., in the treatmentof wounds and other tissue defects such as cosmetic defects as well ashuman and animal feed supplements. For example, growth factors are knownto play an important role in the wound healing process. At least oneactivity of these growth factors is imparted to the conditioned cellmedia or cell extracts through the metabolism processes of thedisclosure.

Cellular cytokines and growth factors are involved in a number ofcritical cellular processes including cell proliferation, adhesion,morphologic appearance, differentiation, migration, inflammatoryresponses, angiogenesis, and cell death. Studies have demonstrated thathypoxic stress and injury to cells induce responses including increasedlevels of mRNA and proteins corresponding to growth factors such as PDGF(platelet-derived growth factor), VEGF (vascular endothelial growthfactor), FGF (fibroblast growth factor), and IGF (insulin-like growthfactor) (Gonzalez-Rubio, M. et al., 1996, Kidney Int. 50(1):164-73;Abramovitch, R. et al., 1997, Int J. Exp. Pathol. 78(2):57-70; Stein, I.et al., 1995, Mol Cell Biol. 15(10):5363-8; Yang, W. et al., 1997, FEBSLett. 403(2):139-42; West, N. R. et al., 1995, J. Neurosci. Res.40(5):647-59).

Growth factors, such as transforming growth factor-β, also known asTGF-β, are induced by certain stress proteins during wound healing. Twoknown stress proteins are GRP78 and HSP90. These proteins stabilizecellular structures and render the cells resistant to adverseconditions. The TGF-β family of dimeric proteins includes TGF-β1,TGF-β2, and TGF-β3 and regulates the growth and differentiation of manycell types. Furthermore, this family of proteins exhibits a range ofbiological effects, stimulating the growth of some cell types (Noda etal., 1989, Endocrinology 124:2991-2995) and inhibiting the growth ofother cell types (Goey et al., 1989, J. Immunol. 143:877-880; Pietenpolet al., 1990, Proc. Natl. Acad. Sci. USA 87:3758-3762). TGF-β has alsobeen shown to increase the expression of extracellular matrix proteinsincluding collagen and fibronectin (Ignotz et al., 1986, J. Biol. Chem.261:4337-4345) and to accelerate the healing of wounds (Mustoe et al.,1987, Science 237:1333-1335).

Another such growth factor is PDGF. PDGF was originally found to be apotent mitogen for mesenchymal-derived cells (Ross R. et al., 1974,Proc. Natl. Acad. Sci. USA 71(4):1207-1210; Kohler N. et al., 1974, Exp.Cell Res. 87:297-301). Further studies have shown that PDGF increasesthe rate of cellularity and granulation in tissue formation. Woundstreated with PDGF have the appearance of an early stage inflammatoryresponse including an increase in neutrophils and macrophage cell typesat the wound site. These wounds also show enhanced fibroblast function(Pierce, G. F. et al., 1988, J. Exp. Med. 167:974-987). Both PDGF andTGF-β have been shown to increase collagen formation, DNA content, andprotein levels in animal studies (Grotendorst, G. R. et al., 1985, J.Clin. Invest. 76:2323-2329; Sporn, M. B. et al., 1983, Science (WashD.C.) 219:1329). PDGF has been shown to be effective in the treatment ofhuman wounds. In human wounds, PDGF-AA expression is increased withinpressure ulcers undergoing healing. The increase of PDGF-AA correspondsto an increase in activated fibroblasts, extracellular matrixdeposition, and active vascularization of the wound. Furthermore, suchan increase in PDGF-AA is not seen in chronic non-healing wounds(Principles of Tissue Engineering, R. Lanza et al. (eds.), pp. 133-141(R.G. Landes Co. TX 1997). A number of other growth factors having theability to induce angiogenesis and wound healing include VEGF, KGF andbasic FGF.

A. Growth Media

Growth media formulations for cell culture are well known in theliterature and many are commercially available.

Preconditioned media ingredients include, but are not limited to thosedescribed below. Additionally, the concentration of the ingredients iswell known to one of ordinary skill in the art. See, for example,Methods For Preparation Of Media, Supplements and Substrate forSerum-free Animal Cell Cultures, supra. The ingredients includeamino-acids (both D and/or L-amino acids) such as glutamine, alanine,arginine, asparagine, cysteine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine and their derivatives; acidsoluble subgroups such as thiamine, ascorbic acid, ferric compounds,ferrous compounds, purines, glutathione and monobasic sodium phosphates.

Additional ingredients include sugars, deoxyribose, ribose, nucleosides,water soluble vitamins, riboflavin, salts, trace metals, lipids, acetatesalts, phosphate salts, HEPES, phenol red, pyruvate salts and buffers.

Other ingredients often used in media formulations include fat solublevitamins (including A, D, E and K) steroids and their derivatives,cholesterol, fatty acids and lipids Tween 80, 2-mercaptoethanolpyramidines as well as a variety of supplements including serum (fetal,horse, calf, etc.), proteins (insulin, transferrin, growth factors,hormones, etc.) antibiotics (gentamicin, penicillin, streptomycin,amphotericin B, etc.) whole egg ultra filtrate, and attachment factors(fibronectins, vitronectins, collagens, laminins, tenascins, etc.).

The media may or may not need to be supplemented with growth factors andother proteins such as attachment factors since many of the cellconstructs, particularly the three-dimensional cell and tissue cultureconstructs described in this application themselves elaborate suchgrowth and attachment factors and other products into the media.

B. Cell Cultures

1. The Cells

Any cells may be used in the cell cultures. For example, the cells maybe stromal cells, parenchymal cells, mesenchymal stem cells (lineagecommitted or uncommitted progenitor cells), liver reserve cells, neuralstem cells, pancreatic stem cells, and/or embryonic stem cells. Thecells may include, but are not limited to, bone marrow, skin, liver,pancreas, kidney, neurological tissue, adrenal gland, mucosalepithelium, and smooth muscle, to name but a few. The fibroblasts andfibroblast-like cells and other cells and/or elements that comprise thestroma may be fetal or adult in origin, and may be derived fromconvenient sources such as skin, liver, pancreas, mucosa, arteries,veins, umbilical cord, and placental tissues, etc. Such tissues and/ororgans can be obtained by appropriate biopsy or upon autopsy. In fact,cadaver organs may be used to provide a generous supply of stromal cellsand elements.

Embryonic stem cells and/or other elements that comprise the stroma maybe isolated using methods known in the art. For instance, recently humanembryonic stem cell populations and methods for isolating and usingthese cells have been reported in Keller et al., Nature Med., 5:151-152(1999), Smith Curr. Biol. 8:R802-804 (1998); isolated from primordialgerm cells, Shamblatt et al., PNAS 95:13726-1373 (1998), isolated fromblastocytes, Thomason et al., Science 282:1145-1147 (1988). Theisolation and culture of mesenchymal stem cells are known in the art.See Mackay et al., Tissue Eng. 4:415-428 (1988); William et al., AmSurg. 65:22-26 (1999). Inoculation of these cells is described infra, inSection 5.3. Likewise, neural stem cells may be isolated in the mannerdescribed in Flax et al., Nature Biotechnol., 16:1033-1039 (1998); andFrisen et al., Cell. Mol. Life. Sci., 54:935-945 (1998).

The cells may be cultured in any manner known in the art including inmonolayer, beads or in three-dimensions and by any means (i.e., culturedish, roller bottle, a continuous flow system, etc.). Methods of celland tissue culturing are well known in the art, and are described, forexample, in Cell & Tissue Culture: Laboratory Procedures, supra,Freshney (1987), Culture of Animal Cells: A Manual of Basic Techniques,infra.

In general, the cell lines utilized are carefully screened for human andanimal pathogens. Depending upon the application, such screening may beof critical importance where only pathogen free cells are acceptable(e.g., for wound healing, food additives, etc.) Methods of screening forpathogens are well known in the art. The cell type, whether cultured intwo-dimensions or three-dimensions, will affect the properties of theconditioned medium. In some embodiments, the cells are cultured in twodimensions. In other embodiments, the cells are cultured in threedimensions.

a. Genetically Engineered Cells

In some embodiments, the cells can act as vehicles for introducing geneproducts into the media or extract that promote the repair and/orregeneration of tissue defects, for example. The cells can begenetically engineered to express, for example, inflammatory mediators,such as IL-6, IL-8 and G-CSF. The cells could also or alternatively begenetically engineering to express anti-inflammatory factors, e.g.,anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc.

In another embodiment, the cells can be genetically engineered toexpress a gene into the media or extract which would exert a therapeuticeffect, e.g., in the production of TGF-β3 to stimulate cartilageproduction, or other factors such as BMP-13 to promote chondrogenesis orstimulatory factors that promote migration of stromal cells and/ormatrix deposition. Since the constructs comprise eukaryotic cells, thegene product will be properly expressed and processed to form an activeproduct. Preferably, the expression control elements used should allowfor the regulated expression of the gene so that the product can beover-synthesized in culture. The transcriptional promoter chosen,generally, and promoter elements specifically, depend, in part, upon thetype of tissue and cells cultured. Cells and tissues which are capableof secreting proteins are preferable (e.g., those having abundant roughendoplasmic reticulum and Golgi complex organelles). The over-producedgene product will then be secreted by the engineered cell into theconditioned media.

The cells may be genetically engineered to regulate one or more genes;or the regulation of gene expression may be transient or long-term; orthe gene activity may be non-inducible or inducible.

The cells can also be genetically engineered to “knock out” expressionof factors that promote inflammation. Negative modulatory techniques forthe reduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation”, as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to the cell can be reduced or knocked out using a number oftechniques, for example, expression may be inhibited by inactivating thegene completely (commonly termed “knockout”) using standard homologousrecombination techniques. Usually, an exon encoding an important regionof the protein (or an exon 5′ to that region) is interrupted by apositive selectable marker (for example neo), preventing the productionof normal mRNA from the target gene and resulting in inactivation of thegene. A gene may also be inactivated by creating a deletion or aninactivating insertion in part of a gene, or by deleting the entiregene. By using a construct with two regions of homology to the targetgene that are far apart in the genome, the sequences intervening the tworegions can be deleted. Mombaerts et al., 1991, Proc. Nat. Acad. Sci.U.S.A. 88:3084-3087. Alternatively, a gene may also be inactivated bydeletion of upstream or downstream expression elements.

Antisense and ribozyme molecules which inhibit expression of the targetgene can also be used to reduce the level of target gene activity. Forexample, antisense RNA molecules which inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Furthermore, appropriateribozyme molecules can be designed as described, e.g., by Haseloff etal., 1988, Nature 334:585-591; Zaug et al., 1984, Science 224:574-578;and Zaug and Cech, 1986, Science 231:470-475. Still further, triplehelix molecules can be utilized in reducing the level of target geneactivity. These techniques are described in detail by L. G. Davis etal., eds, Basic Methods in Molecular Biology, 2nd ed., Appleton & Lange,Norwalk, Conn. 1994.

Methods that may be useful to genetically engineer the cells arewell-known in the art and are further detailed in co-owned U.S. Pat.Nos. 4,963,489 and 5,785,964, the disclosures of which are incorporatedherein by reference. For example, a recombinant DNA construct or vectorcontaining an exogenous nucleic acid, e.g., encoding a gene product ofinterest, may be constructed and used to transform or transfect thestromal cells. Such transformed or transfected cells that carry theexogenous nucleic acid, and that are capable of expressing said nucleicacid, are selected and clonally expanded in the three-dimensionalconstructs of this disclosure.

Methods for preparing DNA constructs containing the gene of interest,for transforming or transfecting cells, and for selecting cells carryingand expressing the gene of interest are well-known in the art. See, forexample, the techniques described in Maniatis et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols inMolecular Biology, Greene Publishing Associates & Wiley Interscience,N.Y.; and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The cells can be engineered using any of a variety of vectors including,but not limited to, integrating viral vectors, e.g., retrovirus vectoror adeno-associated viral vectors; or non-integrating replicatingvectors, e.g., papilloma virus vectors, SV40 vectors, adenoviralvectors; or replication-defective viral vectors. Where transientexpression is desired, non-integrating vectors and replication defectivevectors may be preferred, since either inducible or constitutivepromoters can be used in these systems to control expression of the geneof interest. Alternatively, integrating vectors can be used to obtaintransient expression, provided the gene of interest is controlled by aninducible promoter. Other methods of introducing DNA into cells includethe use of liposomes, lipofection, electroporation, a particle gun, orby direct DNA injection.

The cells are preferably transformed or transfected with a nucleic acid,e.g., DNA, controlled by, i.e., in operative association with, one ormore appropriate expression control elements such as promoter orenhancer sequences, transcription terminators, polyadenylation sites,among others, and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow in enriched mediaand then switched to selective media. The selectable marker in theforeign DNA confers resistance to the selection and allows cells tostably integrate the foreign DNA as, for example, on a plasmid, intotheir chromosomes and grow to form foci which, in turn, can be clonedand expanded into cell lines. This method can be advantageously used toengineer cell lines which express the gene product into the media.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include but are not limited to the CMVpromoter/enhancer, SV40, papillomavirus, Epstein-Barr virus, elastingene promoter and β-globin. Preferably, the control elements used tocontrol expression of the gene of interest should allow for theregulated expression of the gene so that the product is synthesized onlywhen needed in vivo. If transient expression is desired, constitutivepromoters are preferably used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary.Inducible promoters can be built into integrating and/or replicatingvectors. For example, inducible promoters include, but are not limitedto, metallothionien and heat shock protein.

According to one embodiment, the inducible promoters used for expressingexogenous genes of interest are those that are the native promoters ofthose regulatory proteins as disclosed herein that are induced as aresult of cyropreservation and subsequent thawing. For example, thepromoter of TGF-β, VEGF, or various known heat shock proteins can beused as the expression control element, i.e., can be operatively linkedto an exogenous gene of interest in order to express a desired geneproduct in the tissue constructs conditioning the cell media.

A variety of methods may be used to obtain the constitutive or transientexpression of gene products engineered into the cells. For example, thetranskaryotic implantation technique described by Seldon et al., 1987,Science 236:714-718 can be used. “Transkaryotic”, as used herein,suggests that the nuclei of the implanted cells have been altered by theaddition of DNA sequences by stable or transient transfection.Preferably, the cells are engineered to express such gene productstransiently and/or under inducible control during the post-operativerecovery period, or as a chimeric fusion protein anchored to the stromalcells, for example, as a chimeric molecule composed of an intracellularand/or transmembrane domain of a receptor or receptor-like molecule,fused to the gene product as the extracellular domain.

Furthermore, it may be desirable to prepare a construct having anextracellular matrix containing a foreign gene product, growth factor,regulatory factor, etc., which is then found in the conditioned media.This embodiment is based on the discovery that, during the growth ofhuman stromal cells on a three-dimensional support framework, the cellssynthesize and deposit on the framework a human extracellular matrix asproduced in normal human tissue. The extracellular matrix is secretedlocally by cells and not only binds cells and tissue together but alsoinfluences the development and behavior of the cells it contacts. Theextracellular matrix contains various connective tissue proteins, e.g.,fiber-forming proteins interwoven in a hydrated gel composed of anetwork of glycosaminoglycans chains. The glycosaminoglycans are aheterogeneous group of long, negatively charged polysaccharide chains,which (except for hyaluronic acid) are covalently linked to protein toform proteoglycans molecules. According to this embodiment, the stromalcells may be genetically engineered to express a desired gene product,or altered forms of a gene product, which will be present in theextracellular matrix and ultimately the cell medium.

2. Culturing the Cells

In some embodiments, the cells are grown in two-dimensional cellcultures. In other embodiments, the cells are grown in three-dimensionalcell cultures. See, e.g., U.S. Pat. No. 6,372,494, herein incorporatedby reference in its entirety. The stromal cells used in the cellcultures preferably comprise fibroblasts, mesenchymal stem cells, liverreserve cells, neural stem cells, pancreatic stem cells, and/orembryonic stem cells with or without additional cells and/or elementsdescribed more fully herein.

The cells can be cultured by any means known in the art. Preferably, thecells are cultured in an environment which enables aseptic processingand handling. Conventional means of cell and tissue culture have beenlimited by the need for human supervision and control of the media. Thislimits the amount of cells and tissue that can be cultured at a singletime and consequently the volume of conditioned cell media that can beobtained at a single time. For this reason, it is preferred that themedia be conditioned in a manner allowing for large scale growth(yielding large scale conditioned media) using, for example, anapparatus for aseptic large scale culturing like that described inco-owned U.S. Pat. No. 5,763,267 (the '267 patent) which is incorporatedby reference herein in its entirety for all purposes. Using the asepticclosed system described in the '267 patent, preconditioned culture mediais transported from a fluid reservoir to an inlet manifold and evenlydistributed to the cultures in a continuous flow system and is useful inculturing three-dimensional cell and tissue cultures, such asDermagraft® for example. In particular, the apparatus described in the'267 patent includes a plurality of flexible or semi-flexible treatmentchambers comprising one or more individual culture pockets, a pluralityof rigid spacers, an inlet fluid manifold, an outlet fluid manifold, afluid reservoir, and a means for transporting fluid within the system.

During treatment, liquid medium is transported from the fluid reservoirto the inlet manifold, which in turn evenly distributes the media toeach of the connected treatment chambers and internal culture pockets.An outlet fluid manifold is also provided to ensure that each treatmentchamber is evenly filled and to ensure that any air bubbles formedduring treatment are removed from the treatment chambers. The treatmentchambers are flexible or semi-flexible so as to provide for easyend-user handling during rinsing and application of the culturedtransplants. Due to the flexibility of the treatment chambers, rigidspacers are also provided which ensure even fluid distribution withinthe chambers during treatment.

In another embodiment, the tissue is cultivated in an apparatus foraseptic growth of tissue cultures as described in U.S. Pat. No.5,843,766 (the '766 patent) incorporated herein in its entirety for allpurposes. The '766 patent discloses a tissue culture chamber in whichthe chamber is a casing that provides for growth of tissue that can begrown, preserved in frozen form, and shipped to the end user in the sameaseptic container. The tissue culture chamber includes a casingcomprising a substrate within the casing designed to facilitatethree-dimensional tissue growth on the surface of the substrate. Thecasing includes an inlet and an outlet port which assist the inflow andoutflow of medium. The casing also includes at least one flowdistributor. In one embodiment, the flow distributor is a baffle, whichis used to distribute the flow of the medium within the chamber tocreate a continuous, uniform piece of tissue. In a second embodiment,the flow distributor is a combination of deflector plates, distributionchannels, and a flow channel. In each embodiment, the casing furtherincludes a seal so as to ensure an aseptic environment inside thechamber during tissue growth and storage.

Fibroblasts will support the growth of many different cells and tissuesin the three-dimensional culture system, and, therefore, can beinoculated onto the matrix to form a “generic” stromal support matrixfor culturing any of a variety of cells and tissues. However, in certaininstances, it may be preferable to use a “specific” rather than“generic” stromal support matrix, in which case stromal cells andelements can be obtained from a particular tissue, organ, or individual.Moreover, fibroblasts and other stromal cells and/or elements may bederived from the same type of tissue to be cultured in thethree-dimensional system. This might be advantageous when culturingtissues in which specialized stromal cells may play particularstructural/functional roles; e.g., smooth muscle cells of arteries,glial cells of neurological tissue, Kupffer cells of liver, etc.

Once inoculated onto the three-dimensional support, the stromal cellswill proliferate on the framework and deposit the connective tissueproteins naturally secreted by the stromal cells such as growth factors,regulatory factors and extracellular matrix proteins. The stromal cellsand their naturally secreted connective tissue proteins substantiallyenvelop the framework thus forming the living stromal tissue which willsupport the growth of tissue-specific cells inoculated into thethree-dimensional culture system of the disclosure. In fact, wheninoculated with the tissue-specific cells, the three-dimensional stromaltissue will sustain active proliferation of the culture for long periodsof time. Importantly, because openings in the mesh permit the exit ofstromal cells in culture, confluent stromal cultures do not exhibitcontact inhibition, and the stromal cells continue to grow, divide, andremain functionally active.

Growth and regulatory factors are elaborated by the stromal tissue intothe media. Growth factors (for example, but not limited to, αFGF, βFGF,insulin growth factor or TGF-betas), or natural or modified bloodproducts or other bioactive biological molecules (for example, but notlimited to, hyaluronic acid or hormones), enhance the colonization ofthe three-dimensional framework or scaffolding and condition the culturemedia.

The extent to which the stromal cells are grown prior to use of thecultures in vivo may vary depending on the type of tissue to be grown inthree-dimensional tissue culture. The living stromal tissues whichcondition the medium may be used as corrective structures by implantingthem in vivo. Alternatively, the living stromal tissues may beinoculated with another cell type and implanted in vivo. The stromalcells may be genetically engineered to adjust the level of proteinproducts secreted into the culture medium to improve the concentrationof recovered product obtained from the conditioned medium. For example,stromal cells may be genetically engineered to improve concentration ofone or more or the following products obtained from the conditionedmedium. anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF,anti-IL-1, anti-IL-2, etc. Alternatively, the stromal cells may begenetically engineered to “knock out” expression of native gene productsthat promote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or “knock out”expression of MHC in order to lower the risk of rejection.

Growth of the stromal cells in three-dimensions will sustain activeproliferation of both the stromal and tissue-specific cells in culturefor much longer time periods than will monolayer systems. Moreover, thethree-dimensional system supports the maturation, differentiation, andsegregation of cells in culture in vitro to form components of adulttissues analogous to counterparts found in vivo and secure proteins intothe conditional medium more closely resembling physiological ratios.

a. Establishment of Three-Dimensional Stromal Tissue

The three-dimensional support or framework may be of any material and/orshape that: (a) allows cells to attach to it (or can be modified toallow cells to attach to it); and (b) allows cells to grow in more thanone layer. A number of different materials may be used to form theframework, including but not limited to: non-biodegradable materials,e.g., nylon (polyamides), dacron (polyesters), polystyrene,polypropylene, polyacrylates, polyvinyl compounds (e.g.,polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE;teflon), thermanox (TPX), nitrocellulose, cotton; and biodegradablematerials, e.g., polyglycolic acid (PGA), collagen, collagen sponges,cat gut sutures, cellulose, gelatin, dextran, polyalkanoates, etc. Anyof these materials may be woven braided, knitted, etc., into a mesh, forexample, to form the three-dimensional framework. The framework, in turncan be fashioned into any shape desired as the corrective structure,e.g., tubes, ropes, filaments, etc. Certain materials, such as nylon,polystyrene, etc., are poor substrates for cellular attachment. Whenthese materials are used as the three-dimensional framework, it isadvisable to pre-treat the framework prior to inoculation of stromalcells in order to enhance the attachment of stromal cells to thesupport. For example, prior to inoculation with stromal cells, nylonframeworks could be treated with 0.1M acetic acid, and incubated inpolylysine, FBS, and/or collagen to coat the nylon. Polystyrene could besimilarly treated using sulfuric acid.

When the cultures are to be implanted in vivo, it may be preferable touse biodegradable matrices such as polyglycolic acid, collagen, collagensponges, woven collagen, catgut suture material, gelatin, polylacticacid, or polyglycolic acid and copolymers thereof, for example. Wherethe cultures are to be maintained for long periods of time orcryopreserved, non-degradable materials such as nylon, dacron,polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc., may bepreferred. A convenient nylon mesh which could be used in accordancewith the disclosure is NITEX®, a nylon filtration mesh having an averagepore size of 210 μm and an average nylon fiber diameter of 90 μm(#3-210/36, Tetko, Inc., N.Y.).

Stromal cells comprising fibroblasts, mesenchymal stem cells, liverreserve cells, neural stem cells, pancreatic stem cells and/or embryonicstem cells with or without other cells and elements described below, areinoculated onto the framework. Also, cells found in loose connectivetissue may be inoculated onto the three-dimensional support along withfibroblasts. Such cells include but are not limited to smooth musclecells, endothelial cells, pericytes, macrophages, monocytes, plasmacells, mast cells, adipocytes, etc. As previously explained, fetalfibroblasts can be used to form a “generic” three-dimensional stromalmatrix that will support the growth of a variety of different cellsand/or tissues. However, a “specific” stromal tissue may be prepared byinoculating the three-dimensional framework with fibroblasts derivedfrom the same type of tissue to be cultured and/or from a particularindividual who is later to receive the cells and/or tissues grown inculture in accordance with the three-dimensional system.

Thus, in one embodiment, stromal cells which are specialized for theparticular tissue may be cultured. For example, stromal cells ofhematopoietic tissue, including but not limited to fibroblasts,endothelial cells, macrophages/monocytes, adipocytes and reticularcells, could be used to form the three-dimensional subconfluent stromafor the long term culture of bone marrow in vitro. Hematopoietic stromalcells may be readily obtained from the “buffy coat” formed in bonemarrow suspensions by centrifugation at low forces, e.g., 3000×g. In thestromal layer that makes up the inner wall of arteries, a highproportion of undifferentiated smooth muscle cells can be added toprovide the protein elastic. Stromal cells of liver may includefibroblasts, Kupffer cells, and vascular and bile duct endothelialcells. Similarly, glial cells could be used as the stroma to support theproliferation of neurological cells and tissues; glial cells for thispurpose can be obtained by trypsinization or collagenase digestion ofembryonic or adult brain (Ponten and Westermark, 1980, in Federof, S.Hertz, L., eds, “Advances in Cellular Neurobiology,” Vol. 1, New York,Academic Press, pp. 209-227). The growth of cells in thethree-dimensional stromal cell culture may be further enhanced by addingto the framework, or coating the support with proteins (e.g., collagens,elastic fibers, reticular fibers) glycoproteins, glycosaminoglycans(e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate,dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/orother materials.

Further, mesenchymal stem cells (lineage committed or uncommittedprogenitor cells) are advantageous “stromal” cells for inoculation ontothe framework. The cells may differentiate into osteocytes, fibroblastsof the tendons and ligaments, marrow stromal cells, adipocytes and othercells of connective tissue, chondrocytes, depending on endogens orsupplemented growth and regulatory factors and other factors includingprostaglandin, interleukins and naturally—occurring chalones whichregulate proliferation and/or differentiation.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase etc. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, the use of grinders, blenders, sieves, homogenizers,pressure cells, or insonators to name but a few. For a review of tissuedisaggregation techniques, see Freshney, Culture of Animal Cells: AManual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch.9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells: A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

The isolation of fibroblasts may, for example, be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalmatrix (see, Naughton et al., 1987, J. Med. 18 (3 and 4) 219-250).Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10⁶ to 5×10⁷ cells/ml, will resultin the establishment of the three-dimensional stromal tissue in shorterperiods of time.

After inoculation of the stromal cells, the three-dimensional frameworkshould be incubated in an appropriate nutrient medium. As previouslymentioned, many commercially available media such as RPMI 1640,Fisher's, Iscove's, McCoy's, and the like may be suitable for use. It isimportant that the three-dimensional stromal cell cultures be suspendedor floated in the medium during the incubation period in order tomaximize proliferative activity. The culture is “fed” periodically andthe conditioned media is recovered and processed as described below inSections 5.6 and 5.7. Thus, depending upon the tissue to be cultured andthe collagen types desired, the appropriate stromal cell(s) may beselected to inoculate the three-dimensional matrix.

During incubation of the three-dimensional stromal cell cultures,proliferating cells may be released from the matrix. These releasedcells may stick to the walls of the culture vessel where they maycontinue to proliferate and form a confluent monolayer. This should beprevented or minimized, for example, by removal of the released cellsduring feeding, or by transferring the three-dimensional stromal cultureto a new culture vessel. The presence of a confluent monolayer in thevessel will “shut down” the growth of cells in the three-dimensionalmatrix and/or culture. Removal of the confluent monolayer or transfer ofthe culture to fresh media in a new vessel will restore proliferativeactivity of the three-dimensional culture system. It should be notedthat the conditioned media is processed, if necessary, so that it doesnot contain any whole cells (unless whole cells are used for a specificapplication). Such removal or transfers should be done in any culturevessel which has a stromal monolayer exceeding 25% confluency.Alternatively, the culture system could be agitated to prevent thereleased cells from sticking, or instead of periodically feeding thecultures, the culture system could be set up so that fresh mediacontinuously flows through the system. The flow rate could be adjustedto both maximize proliferation within the three-dimensional culture, andto wash out and remove cells released from the culture, so that theywill not stick to the walls of the vessel and grow to confluence.

Other cells, such as parenchymal cells, may be inoculated and grown onthe three-dimensional living stromal tissue.

b. Innoculation of Additional Cells into the Cell Cultures

Once the cell culture has reached a particular degree of growth,additional cells may be inoculated into the cell culture and culturedalong with the cells of the cell culture. The additional cells are grownon the living tissue in vitro to form a cultured counterpart of thenative tissue and condition the media by elaborating extracellularproducts into the media at ratios resembling physiological levels. Ahigh concentration of cells in the inoculum will advantageously resultin increased proliferation in culture much sooner than will lowconcentrations.

The cells chosen for inoculation will depend upon the tissue to becultured, which may include, but is not limited to, bone marrow, skin,liver, pancreas, kidney, neurological tissue, adrenal gland, mucosalepithelium, and smooth muscle, to name but a few. Such cells withelaborate characteristic extracellular proteins such as certain growthfactors into the media resulting in media optimized for certain tissuespecific applications.

For example, and not by way of limitation, a variety of epithelial cellscan be cultured on the living tissue. Examples of such epithelial cellsinclude, but are not limited to, keratinocytes, oral mucosa andgastrointestinal (G.I.) tract cells. Such epithelial cells may beisolated by enzymatic treatment of the tissue according to methods knownin the art, followed by expansion of these cells in culture andapplication of epithelial cells to, for example, the three-dimensionalstromal support cell matrix. The presence of the stromal supportprovides growth factors and other proteins which promote normal divisionand differentiation of epithelial cells.

In general, this inoculum should include the “stem” cell (also calledthe “reserve” cell) for that tissue; i.e., those cells which generatenew cells that will mature into the specialized cells that form thevarious components of the tissue.

The parenchymal or other surface layer cells used in the inoculum may beobtained from cell suspensions prepared by disaggregating the desiredtissue using standard techniques described above for obtaining stromalcells. The entire cellular suspension itself could be used to inoculatethe three-dimensional living stromal tissue. As a result, theregenerative cells contained within the homogenate will proliferate,mature, and differentiate properly on the matrix, whereasnon-regenerative cells will not. Alternatively, particular cell typesmay be isolated from appropriate fractions of the cellular suspensionusing standard techniques described above for fractionating stromalcells. Where the “stem” cells or “reserve” cells can be readilyisolated, these may be used to preferentially inoculate thethree-dimensional stromal support. For example, when culturing bonemarrow, the stroma may be inoculated with bone marrow cells, eitherfresh or derived from a cryopreserved sample. When culturing skin, thethree-dimensional stroma may be inoculated with melanocytes andkeratinocytes. When culturing liver, the three-dimensional stroma may beinoculated with hepatocytes. When culturing pancreas, thethree-dimensional stroma may be inoculated with pancreatic endocrinecells. For a review of methods which may be utilized to obtainparenchymal cells from various tissues, see, Freshney, Culture of AnimalCells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York,1987, Ch. 20, pp. 257-288.

In fact, different proportions of the various types of collagendeposited on the stromal matrix prior to inoculation can affect thegrowth of the later-inoculated tissue-specific cells. For example, foroptimal growth of hematopoietic cells, the matrix should preferablycontain collagen types III, IV and I in an approximate ratio of 6:3:1 inthe initial matrix. For three dimensional skin culture systems, collagentypes I and III are preferably deposited in the initial matrix. Theproportions of collagen types deposited can be manipulated or enhancedby selecting fibroblasts which elaborate the appropriate collagen type.This can be accomplished using monoclonal antibodies of an appropriateisotype or subclass that is capable of activating complement, and whichdefine particular collagen types. These antibodies and complement can beused to negatively select the fibroblasts which express the desiredcollagen type. Alternatively, the stromal cells used to inoculate thematrix can be a mixture of cells which synthesize the appropriatecollagen type desired. The distribution and origins of various types ofcollagen is shown in Table I.

TABLE 1 DISTRIBUTIONS AND ORIGINS OF VARIOUS TYPES OF COLLAGEN CollagenType Principal Tissue Distribution Cells of Origin I Loose and denseordinary Fibroblasts and reticular connective tissue; collagen fiberscells; smooth muscle cells Fibrocartilage Bone Osteoblast DentinOdontoblasts II Hyaline and elastic cartilage Chondrocytes Vitreous bodyof eye Retinal cells III Loose connective tissue; Fibroblasts andreticular reticular fibers cells Papillary layer of dermis Blood vesselsSmooth muscle cells; endothelial cells IV Basement membranes Epithelialand endothelial cells Lens capsule of eye Lens fibers V Fetal membranes;placenta Fibroblast Basement membranes Bone Smooth muscle Smooth musclecells Fibroblasts VI Connective Tissue VII Epithelial basementmembranes, Fibroblasts, keratinocytes anchoring fibrils Cornea Cornealfibroblasts VIII Cartilage IX Hypertrophic cartilage X CartilageFibroblasts XI Papillary dermis Fibroblasts XII Reticular dermisFibroblasts XIV, P 170 bullous pemphigoid Keratinocytes undulin antigen

During incubation, the cell culture system should be suspended orfloated in the nutrient medium. Cultures should be fed with fresh mediaperiodically. Again, care should be taken to prevent cells released fromthe culture from sticking to the walls of the vessel where they couldproliferate and form a confluent monolayer. The release of cells fromthe culture appears to occur more readily when culturing diffuse tissuesas opposed to structured tissues. For example, the skin culture ishistologically and morphologically normal; the distinct dermal andepidermal layers do not release cells into the surrounding media. Bycontrast, the bone marrow cultures release mature non-adherent cellsinto the medium much the way such cells are released in marrow in vivo.As previously explained, should the released cells stick to the culturevessel and form a confluent monolayer, the proliferation of thethree-dimensional culture will be “shut down”. This can be avoided byremoval of released cells during feeding, transfer of the culture to anew vessel, by agitation of the culture to prevent sticking of releasedcells to the vessel wall, or by the continuous flow of fresh media at arate sufficient to replenish nutrients in the culture and removereleased cells. As previously mentioned, the conditioned media isprocessed, if necessary, so that it is free of whole cells and cellulardebris.

The growth and activity of cells in culture can be affected by a varietyof growth factors such as insulin, growth hormone, somatomedins, colonystimulating factors, erythropoietin, epidermal growth factor, hepaticerythropoietic factor (hepatopoietin), and liver-cell growth factor.Other factors which regulate proliferation and/or differentiationinclude prostaglandins, interleukins, and naturally-occurring chalones.

C. Conditioned Growth Media and Cell Extract

1. Recovering the Conditioned Growth Media or Cell Extract from the CellCultures

The resultant conditioned growth medium may be separated from the cellcultures by any means known to the skilled artisan. In one embodiment,the conditioned growth medium is pumped out of the cell culture systemand processed for use. In some embodiments, the conditioned growthmedium is recovered once the cell cultures have conditioned the growthmedium to a sufficient extent (i.e., once the medium is conditioned sothat extracellular proteins such as growth factors have reacheddesirable levels in the medium). Preferably, the conditioned growthmedium is recovered at the later stages of growth of the tissue when thelevel of certain growth factors and connective tissue protein secretionis at its highest level (See FIG. 1). In a preferred embodiment, theconditioned growth medium is recovered after exposure of the growthmedium to the cells at days 10 through day 14 of culturing.

The cell extract produced by the cell cultures can be isolated from theconditioned growth medium by any means known to the skilled artisan.

2. Processing the Conditioned Growth Media or Cell Extract

Following recovery of the conditioned growth medium or cell extract, itmay be necessary to further process the resulting supernatant. Suchprocessing may include, but are not limited to, concentration by a waterflux filtration device or by defiltration using the methods described inCell & Tissue Culture: Laboratory Procedures, supra, pp 29D:0.1-29D:0.4.

Additionally, the medium may be concentrated 10 to 20 fold using apositive pressure concentration device having a filter with a 10,000 mlcut-off (Amicon, Beverly, Mass.).

Also, the conditioned growth medium or cell extract may be furtherprocessed for product isolation and purification to remove unwantedproteases, for example. The methods used for product isolation andpurification so that optimal biological activity is maintained will bereadily apparent to one of ordinary skill in the art. For example, itmay be desirous to purify a growth factor, regulatory factor, peptidehormone, antibody, etc. Such methods include, but are not limited to,gel chromatography (using matrices such as sephadex) ion exchange, metalchelate affinity chromatography with an insoluble matrix such ascross-linked agarose, HPLC purification and hydrophobic interactionchromatography of the conditioned media. Such techniques are describedin greater detail in Cell & Tissue Culture: Laboratory Procedures,supra. Depending upon the desired application of the conditioned growthmedium or cell extract, and/or products derived thereof, appropriatemeasures must be taken to maintain sterility. Alternatively,sterilization may be necessary and can be accomplished by methods knownto one of ordinary skill in the art, such as, for example, heat and/orfilter sterilization taking care to preserve the desired biologicalactivity.

3. Some Products Found in the Conditioned Growth Media or Cell Extract

Table 2 below lists the concentration of a number of growth factorsdetermined by ELISA (enzyme linked immuno assay) to be in Applicants'conditioned medium which previously supported the growth of the cellsgrown in Dermagraft® tissue culture. It is believed that a cell extractaccording to the present invention would also contain these growthfactors. It should be understood that the following list is not an allinclusive list of factors and is provided solely to further characterizethe conditioned medium by providing the concentration of some of thebiologically active factors present in the medium of the disclosure.

TABLE 2 Growth Factor Concentrations in Conditioned Medium as Measuredby ELISA VEGF 3.2 ng/ml G-CSF 2.3 ng/ml IL-8 0.9 ng/ml IL-8 3.2 ng/mlKGF 1.67 ng/ml TGF-β 0.8 ng/ml

A variety of methods have been utilized to quantify and characterize themajor molecular components secreted by fibroblasts found in thethree-dimensional tissue cultures TransCyte™ and Dermagraft®). The humanmatrix proteins and glycosaminoglycans (GAGs) present in TransCyte™ andDermagraft® include, but are not limited to, collagen I, III,fibronectin, tenascin, decorin, versican betaglycan, syndecan as well asother components (data not shown). These secreted proteins and GAGsserve major structural functions as well as stimulate cell division,migration, adhesion and signal transduction. The deposition ofglycosaminoglycans (deposition volume is dependent on period of growth)and collagen (deposition volume is not dependent on period of growth) inthe three-dimensional growth systems are illustrated in FIG. 1. Thecomponents have been measured by ELISA, Western blot analysis, immunohistochemistry and PCR. For example, some of the components found inTransCyte™ include collagen I, III, and VII (RNA), fibronectin,tenascin, thrombospondin 2, elastin, proteoglycans, decorin, versican aswell as other components (data not shown). Activity of these componentsin tissue development, healing, and normal function have been welldescribed. Additionally, Applicants describe certain effects of thehuman bioengineered matrix on cell function in vitro. For example,Applicants have noted that cell proliferation is increased by addingbioengineered matrix. To study its effects on cell proliferation, matrixwas physically removed from TransCyte™ and Dermagraft® and added invarying dilutions to monolayer cultures of human fibroblasts andkeratinocytes. The results of increased cell proliferation are shown inFIG. 2.

Further, Applicants note the effect of three dimensional conditionedmedium on the preparation and composition of three-dimensional tissueswas examined by measuring the amount of collagen secreted into theextracellular matrix of tissues cultured in the presence of serum-freemedium, medium or three dimensional conditioned medium. The effect ofthree dimensional conditioned medium on the preparation and compositionof three-dimensional tissues was examined by measuring the amount ofcollagen secreted into the extracellular matrix of tissues cultured inthe presence of serum-free medium, medium or three dimensionalconditioned medium. The conditioned medium of the disclosuresignificantly increases collagen deposition of tissue in vitro.

D. Metabolizing the Conditioned Growth Media or Cell Extract

1. The Yeast

The process of metabolizing the conditioned growth media or cell extractcan occur with a variety of microorganisms such as, for example, yeast,bacillus, molds, plant cells and the like. Especially preferred for thepresent disclosure are ferments made using yeast. As used herein, theterm “yeast” is meant to encompass a single yeast cell, multiple yeastcells and/or a culture of yeast cells. The yeast can be of variousfungal families, known to those skilled in the art including, but notlimited to: Neurospora, Ceratostomella, Claviceps, Xylaria, Rosellinia,Helotium, Sclerotinia, Tulostoma, Rhizopogon, Truncocolumella, Mucor,Rhizopus, Entomophthora, Dictostylium, Blastocladia, Synchytrium,Saprolegnia, Peronospora, Albugo, Pythium, Phytophthora, Plasmodiophora,Tuber, Hydnum, Lecanora, Roccella, Pertusaria, Usnea, Evernia, Ramalina,Alectoria, Cladonia, Parmelia, Cetraria, Agaricus, Cantharellus,Omphalotus, Coprinus, Lactarius, Marasmius, Pleurotus, Pholiota,Russula, Lactarius, Stropharia, Entoloma, Lepiotaceae, Corticium,Pellicularia, Tricholoma, Volvaria, Clitocybe, Flammulina,Saccharomyces, Schizosaccharomyces, Saccharomycetaceae, Eurotium,Aspergillus, Thielavia, Peziza, Plectania, Morchella, Wynnea, Helvella,Gyromitra, Phallales, Dictyophera, Mutinus, Clathrus, Pseudocolus,Lycoperdon, Calvatia, Geastrum, Radiigera, Astreus, Nidularia,Gastrocybe, Macowanites, Gastroboletus, Albatrellus, Neolentinus,Nigroporus, Oligoporus, Polyporus, Fistulina, Fomes, Boletus,Fuscoboletinus, Leccinum, Phylloporus, Suillus, Strobilomyces,Boletellus, Tremella, Auricularia, Dacrymyces, Melampsora, Cronartium,Puccinia, Gymnosporangium, Tilletia, Urocystis, Septobasidium,Hygrocybe, Hygrophorus, Hygrotrama, Neohygrophorus, Cortinarius,Gymnopilus, Trichophyton, Microsporum, Monilia, Candida, Cercosporella,Penicillium, Blastomyces, Cercospora, Ustilaginoidea, Tuber cularia,Fusarium, Rhizoctinia, Ozonium, Sclerotium, Geoglossum, or Armillaria.Of particular interest are the fungi belonging to the familySaccharomycetaceae. Of greater interest are the fungi belonging to thegenus Pichia. Of most interest are the fungi belonging to the speciespastoris. In a preferred embodiment, Pichia pastoris is used in thefermentation process. In some embodiments, the yeast is aerobicallygrown.

2. Fermentation Process

The yeast fermentation process can be carried out in a stirred tankbio-reactor. Examples of such bioreactors might include for example,fermentors available from New Brunswick Scientific, Edison N.J. orApplikon Biotechnology Foster City Calif.

The yeast extracts of the present disclosure include cytoplasmic andextra-cellular components of the yeast which include, but are notlimited to, the nutrient broth, cellular protein material, cellularnucleic material, cellular protoplasmic material and/or cell wallcomponents. In some embodiments, the extracts are relatively watersoluble, for example, equal or more than 1-gram of yeast extractsdissolve in 1-gram of water. The extracts may also be soluble inwater/organic solvent mixtures such as, but not limited to, aqueousglycols and aqueous glycerols.

3. Recovering/Processing the Metabolized Conditioned Growth Media orMetabolized Cell Extract

The metabolized conditioned growth media or metabolized cell extract maybe recovered and/or processed according to the procedures describedabove for recovering and processing the conditioned growth media or cellextract. In some embodiments, the processing includes several steps andmay involve the use of techniques, including but not limited tocontinuous flow centrifugation, lenticular dead-end filtration forclarification of bio-mass, and tangential flow filtration with sterileand ultra-filtration capabilities. In one embodiment, the finalprocessing step includes tangential flow filtration of a specificmolecular weight cut-off in order to selectively target bio-activecomponents. See, e.g., “Yeast Protocols” W. Xiao ed. (Humana Press2006), which is incorporated by reference herein.

E. Activity/Characterization of the Metabolized Conditioned Growth Mediaor Metabolized Cell Extract

During the yeast metabolism described above, therapeutic productspresent in the conditioned growth media and cell extract, including butnot limited to enzymes, hormones, cytokines, antigens, antibodies,clotting factors, and regulatory proteins, are metabolized by the yeast.The term metabolized is known to those skilled in the art butessentially means that the yeast begin the process of digesting thecomponents of the conditioned nutrient media or cell extract. Theprocess of metabolizing can render larger molecules as small, morereadily available components that are more useful for the applicationsof the present invention than the unmetabolized conditioned growth mediaor cell extracts. Therapeutic proteins that may be metabolized include,but are not limited to, inflammatory mediators, angiogenic factors,Factor VIII, Factor IX, erythropoietin (EPO), alpha-1 antitrypsin,calcitonin, glucocerebrosidase, human growth hormone and derivatives,low density lipoprotein (LDL), and apolipoprotein E, IL-2 receptor andits antagonists, insulin, globin, immunoglobulins, catalytic antibodies,the interleukins (ILs), insulin-like growth factors, superoxidedismutase, immune responder modifiers, BMPs (bone morphogenic proteins)parathyroid hormone and interferon, nerve growth factors, tissueplasminogen activators, and colony stimulating factors (CSFs). Thesemetabolized components may maintain the activity of their precursors, ormay be transformed to have greater enhanced activity, including thedownregulation or upregulation of various physiological systems thataffect, for example, skin maintenance and condition.

The medium or extract may be further processed to concentrate or reduceone or more metabolized factor or component contained within the mediumor extract, for example, enrichment of a metabolized growth factor usingimmunoaffinity chromatography or, conversely, removal of a lessdesirable component, for any given application as described herein.

In other embodiments, the metabolized conditioned growth media and/ormetabolized cell extract of the present disclosure contains one or morecytoplasmic and extra-cellular components of the yeast which include,but are not limited to, the nutrient broth, cellular protein material,cellular nucleic material, cellular protoplasmic material and/or cellwall components.

III. COMPOSITIONS

In one embodiment, the disclosure provides compositions comprising themetabolized conditioned growth medium or the metabolized cell extract,and an acceptable carrier. In some embodiments, the composition is apersonal care composition.

In some embodiments, the metabolized conditioned growth medium or themetabolized cell extract is present in the composition in an amount ofabout 0.0001% to about 95% by weight of the composition. In otherembodiments, the metabolized conditioned growth medium or themetabolized cell extract is present in the composition in an amount ofabout 0.01% to about 50% by weight of the composition. In otherembodiments, the metabolized conditioned growth medium or themetabolized cell extract is present in the composition in an amount ofabout 5% to about 30% by weight of the composition. In otherembodiments, the metabolized conditioned growth medium or themetabolized cell extract is present in the composition in an amount ofabout 10% to about 20% by weight of the composition. In otherembodiments, the metabolized conditioned growth medium or themetabolized cell extract is present in the composition in an amount ofabout 13% to about 17% by weight of the composition. In otherembodiments, metabolized conditioned growth medium or the metabolizedcell extract is present in the composition in an amount of about 0.01%to about 10% by weight of the composition. In one embodiment, themetabolized conditioned growth medium or the metabolized cell extract ispresent in the composition in an amount of about 10%, about 15%, orabout 20% by weight of the composition.

In other embodiments, the composition further comprises at least one ofwater, a preservative, a surfactant, an emulsifier, a conditioner, anemollient, a wax, an oil, a polymer, a thickener, a fixative, acolorant, a humectant, a moisturizer, a stabilizer, a diluent, a solventand a fragrance. Non-limiting examples of additional ingredients of thecompositions include, but are not limited to, Palmitoyl trippeptide 5,hydrolyzed silk (Sericin), Dipalmitoyl hydroxyproline, Ergothioneine(EGT), Ubiquinone (Coenzyme Q10), Camellia Sinensis (Green Tea) LeafExtract, Tetrahexyldecyl Ascorbate, Tocopheryl Acetate, Rubus Fruticosus(Blackberry) Leaf Extract, Saccharomyces Ferment Filtrate Lysate,Alpha-Arbutin, Gamma Aminobutyric Acid, and/or Hyaluronic FillingSpheres.

In one embodiment, the composition further comprises at least onepreservative. Suitable preservatives include, but are not limited to,acids, alcohols, glycols, parabens, quaternary-nitrogen containingcompounds, isothiazolinones, aldehyde-releasing compounds andhalogenated compounds. Illustrative alcohols include phenoxyethanol,isopropyl alcohol, and benzyl alcohol; illustrative glycols includepropylene, butylene and pentylene glycols; illustrative parabens include(also known as parahydroxybenzioc acids) methyl, propyl and butylparabens; illustrative quaternary nitrogen containing compounds includebenzalkonium chloride, Quartenium 15; illustrative isothiazoles includemethylisothiazoline, methychlorolisothiazoline; illustrative aldehydereleasing agents include DMDM hydantion, imiadolidinyl urea anddiazolidinyl urea; illustrative antioxidants include butylatedhydroxytoluene, tocopherol and illustrative halogenated compoundsinclude triclosan and chlorohexidene digluconate. Examples ofpreservatives useful for the purpose of the present disclosure can befound in Steinberg, D. “Frequency of Use of Preservatives 2007”. Cosmet.Toilet. 117, 41-44 (2002) and, “Preservative Encyclopedia” Cosmet.Toilet. 117, 80-96 (2002). In addition, enzyme preservative systems suchas those described in the article by Ciccognani D. Cosmetic PreservationUsing Enzymes, in “Cosmetic and Drug Microbiology”, Orth D S ed.,Francis & Taylor, Boca Raton, Fla. (2006) can also be effective for usewith the composition of the present disclosure.

In one embodiment, the composition further comprises an activeingredient. Suitable active ingredients include, but are not limited tobotanicals, nutraceuticals, cosmeceuticals, therapeutics,pharmaceuticals, antimicrobials, steroidal hormones, antidandruffagents, anti-acne components, sunscreens, antibiotics, antivirals,antifungals, steroids, analgesics, antitumor drugs, investigationaldrugs or any compounds which would result in a complimentary orsynergistic combination with the factors in the metabolized conditionedmedia or metabolized cell extract.

The compositions may be in the form of tablets, capsules, skin patches,inhalers, eye drops, nose drops, ear drops, suppositories, creams,ointments, injectables, hydrogels and into any other appropriateformulation known to one of skill in the art. For oral administrationthe pharmaceutical compositions may take the form of, for example,tablets or capsules prepared by conventional means with acceptableexcipients or carriers such as binding agents (e.g., pregelatinisedmaize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolae); orwetting agents (e.g., sodium lauryl sulphate). Tablets may be coatedusing methods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with acceptableexcipients or carriers such as suspending agents (e.g., sorbitol syrupcellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate.

In one embodiment, the compositions are in a form suitable for cosmeticapplication including, but not limited to, lotions, ointments, creams,sprays, spritzes, aqueous or aqueous-alcoholic mixture gels, mousses,patches, pads, masks, moistened clothes, wipes, solid sticks, clearsticks, lip sticks, aerosol creams, anhydrous powders, talcs, tonics,oils, emulsions or bath salts.

In one embodiment, the metabolized conditioned growth medium or themetabolized cell extract in the composition is present in a chemicaldelivery vehicle. Chemical delivery vehicles include, but are notlimited to, liposomes, niosomes, sub-micron emulsions, polymericencapsulates, gels, creams, lotions, and combinations thereof. In someembodiments, the metabolized conditioned growth medium or themetabolized cell extract is encapsulated within an encapsulant.

The compositions of the disclosure may be delivered to a subject via avariety of routes using standard procedures well known to those of skillin the art. For example, such delivery may be site-specific, oral,nasal, intravenous, subcutaneous, intradermal, transdermal,intramuscular or intraperitoneal administration. Also, they may beformulated to function as controlled or slow release vehicles.

The metabolized conditioned growth medium and/or metabolized cellextract may be used in any state, i.e., liquid or solid, frozenlyophilized or dried into a powder, as a film for topical woundtreatments and anti-adhesion applications, as an injectable, see PCT WO96/39101, incorporated herein by reference it its entirety.

Alternatively, the conditioned cell medium of the present disclosure maybe formulated with polymerizable or cross-linking hydrogels as describedin U.S. Pat. Nos. 5,709,854; 5,516,532; 5,654,381; and WO 98/52543, eachof which is incorporated herein by reference in its entirety. Examplesof materials which can be used to form a hydrogel include modifiedalginates. Alginate is a carbohydrate polymer isolated from seaweed,which can be cross-linked to form a hydrogel by exposure to a divalentcation such as calcium, as described, for example in WO 94/25080, thedisclosure of which is incorporated herein by reference. Alginate isionically cross-linked in the presence of divalent cations, in water, atroom temperature, to form a hydrogel matrix. As used herein, the term“modified alginates” refers to chemically modified alginates withmodified hydrogel properties.

Additionally, polysaccharides which gel by exposure to monovalentcations, including bacterial polysaccharides, such as gellan gum, andplant polysaccharides, such as carrageenans, may be cross-linked to forma hydrogel using methods analogous to those available for thecross-linking of alginates described above.

Modified hyaluronic acid derivatives are particularly useful. As usedherein, the term “hyaluronic acids” refers to natural and chemicallymodified hyaluronic acids. Modified hyaluronic acids may be designed andsynthesized with preselected chemical modifications to adjust the rateand degree of cross-linking and biodegradation.

Covalently cross-linkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate.

Alternatively, polymers may be utilized which include substituents whichare cross-linked by a radical reaction upon contact with a radicalinitiator. For example, polymers including ethylenically unsaturatedgroups which can be photochemically cross-linked which may be utilized,as disclosed in WO 93/17669, the disclosure of which is incorporatedherein by reference. In this embodiment, water soluble macromers thatinclude at least one water soluble region, a biodegradable region, andat least two free radical-polymerizable regions, are provided. Examplesof these macromers are PEG-oligolactyl-acrylates, wherein the acrylategroups are polymerized using radical initiating systems, such as aneosin dye, or by brief exposure to ultraviolet or visible light.Additionally, water soluble polymers which include cinnamoyl groupswhich may be photochemically cross-linked may be utilized, as disclosedin Matsuda et al., ASAID Trans., 38: 154-157 (1992).

The preferred polymerizable groups are acrylates, diacrylates,oligoacrylates, dimethacrylates, oligomethacrylates, and otherbiologically acceptable photopolymerizable groups. Acrylates are themost preferred active species polymerizable group.

Naturally occurring and synthetic polymers may be modified usingchemical reactions available in the art and described, for example, inMarch, “Advanced Organic Chemistry”, 4^(th) Edition, 1992,Wiley-Interscience Publication, New York.

Polymerization is preferably initiated using photo initiators. Usefulphoto initiators are those which can be used to initiate polymerizationof the macromers without cytotoxicity and within a short time frame,minutes at most and most preferably seconds.

Numerous dyes can be used for photopolymerization. Suitable dyes arewell known to those of skill in the art. Preferred dyes includeerythrosin, phloxime, rose bengal, thonine, camphorquinone, ethyl eosin,eosin, methylene blue, riboflavin, 2,2-dimethyl-2-phenylacetophenone,2-methoxy-2-phenylacetophenone, 2,2-dimethoxy-2-phenyl acetophenone,other acetophenone derivatives, and camphorquinone. Suitable cocatalystsinclude amines such as N-methyl diethanolamine, N,N-dimethylbenzylamine, triethanol amine, trithylamine, dibenzyl amine,N-benzylethanolamine, -isopropyl benzylamine. Triethanolamine is apreferred cocatalyst.

In another embodiment, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure, or alternatively particularmetabolized extracellular matrix proteins elaborated into the media, areused to provide an excellent substance to coat sutures. The metabolized,naturally secreted extracellular matrix provides the conditioned mediawith an activity of type I and type III collagens, fibronectin,terascin, glycosaminologycans, acid and basic FGF, TGF-α and TGF-β, KGF,versican, decorin and various other secreted human dermal matrixproteins. Similarly, the conditioned cell media of the disclosure or theextracellular matrix proteins derived from the conditioned media may beused to coat conventional implantation devices, including vascularprosthesis, in surgical approaches to correct defects in thebody—resulting in superior implantation devices. The implants should bemade of biocompatible, inert materials that replace or substitute forthe defective function and made of either non-biodegradable materials orbiodegradable materials. By coating implantation devices with the mediumcontaining these extracellular proteins, the implant invites propercellular attachments resulting in superior tissue at the implantationsite. Thus, sutures, bandages, and implants coated with conditioned cellmedia, or proteins derived from the media, enhance the recruitment ofcells, such as leukocytes and fibroblasts into the injured area andinduce cell proliferation and differentiation resulting in improvedwound healing.

In another embodiment, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may be formulated with apharmaceutically acceptable carrier as a vehicle for internaladministration. Also, the medium may be further processed to concentrateor reduce one or more factor or component contained within the medium,for example, enrichment of a growth factor or other protein usingimmunoaffinity chromatography or, conversely, removal of a lessdesirable component, for any given application as described herein.

IV. METHODS FOR TREATING OR PREVENTING CONDITIONS

In one embodiment, the disclosure provides methods for preventing ortreating a condition in a subject comprising administering to thesubject a therapeutically effective amount of a composition comprising:an acceptable carrier and a metabolized conditioned growth medium.

In another embodiment, the disclosure provides methods for preventing ortreating a condition in a subject comprising administering to thesubject a therapeutically effective amount of a composition comprising:an acceptable carrier and a metabolized cell extract.

Conditions to be treated include, but are not limited to, skinconditions, including cosmetic defects, congenital defects, hair lossand acquired defects. In some embodiments, the conditions to be treatedinclude, but are not limited to, fine lines and wrinkles; age spots anddyspigmentation; decreased skin texture, tone and elasticity; roughnessand photo damage; decreased ability of skin to regenerate itself;environmental damage; decreased smoothness and tightness of skin; agespots; fine and coarse lines and wrinkles; fine and coarse periocularwrinkles; nasolabial folds; facial fine and coarse lines; decreased skinradiance and evenness; decreased skin firmness; hyperpigmentation; darkspots and/or patches; decreased skin brightness and youthful appearance;photoaged skin; intrinsically and extrinsically aged skin; abnormal skincellular turnover; decreased skin barrier; decrease of skin's ability toretain moisture; brown and red blotchiness; redness; abnormal skinepidermal thickness; reduction of dermal epidermal junction; increasedpore size and number of pores; or a combination thereof.

A. Skin Conditions

In some embodiments, the condition to treat or prevent is a skincondition. In other embodiments, the skin condition is a cosmeticdefect, a congenital defect, hair loss or an acquired defect Skinconditions also include, but are not limited to, fine lines andwrinkles; age spots and dyspigmentation; decreased skin texture, toneand elasticity; roughness and photo damage; decreased ability of skin toregenerate itself; environmental damage; decreased smoothness andtightness of skin; age spots; fine and coarse lines and wrinkles; fineand coarse periocular wrinkles; nasolabial folds; facial fine and coarselines; decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof.

In one non-limiting example, a topical composition as described hereinis applied to the skin. Application of the compositions disclosed hereinrejuvenates sun damaged and aging skin; improves the appearance of finelines and wrinkles; promotes cell renewal; diminishes the appearance ofage spots and dyspigmentation; improves skin tone, texture andelasticity; reduces roughness and photo damage; prevents or reducesenvironmental damage; plumps the skin; brightens the skin; lightens theskin; strengthens the ability of skin to regenerate itself; improves theappearance of age spots; brightens and lightens age spots; improves skinfirmness, elasticity, resiliency; smoothes, tightens, or fills in finelines on the skin; reduces the appearance of dark circles under the eye;improves lip texture or condition; enhances natural lip color; increaseslip volume; promotes epithelialization of post-procedure skin; restoresthe skin's barrier or moisture balance; improves the appearance of agespots; improves the appearance of skin pigmentation, or a combinationthereof.

In certain embodiments, administration of a composition described hereinmay result in at least a 2-fold improvement of one or more symptoms orconditions. Folds improvement of one or more symptoms or conditionsinclude, but are not limited to, 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 75-fold, 100-fold or more, or any number therebetween.In certain embodiments, administration of a composition described hereinmay result in improvement of about 1% to about 100%, about 10% to about90%, about 20% to about 80%, about 30% to about 70%, about 40% to about60%, or about 50%. In other embodiments, administration of a compositiondescribed herein may result in improvement of one or more symptoms orconditions of about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 100%, about 125%, about 150% or more of one or more symptoms orconditions.

The effect of the metabolized conditioned growth media and/ormetabolized cell extract described above to influence human skin can bemeasured in a number of ways known to those skilled in the art. Inparticular, the metabolized conditioned growth media and/or metabolizedcell extract described above can be screened for their effects on skinby employing analytical techniques such as, for example, human genomicmicroarrays on specific skin cells such as keratinocytes or fibroblasts,or by protein expression analysis on individual skin cells, tissuemodels or ex vivo or in vivo skin models. In these testing models,specific genes and or proteins may be up-regulated or down-regulated asa result of the extract treatment. Genes and proteins that are capableof regulating skin conditions are of particular interest in thescreening. Of particular interest for the purpose of the presentdisclosure are genes and proteins related to inflammation, extracellularmatrix expression, melanin regulation, skin moisturization, exfoliationand the like. Of particular interest are proteins related tocyclooxygenase expression, in particular cyclooxygenase-1 and 2 and alsoextracellular matrix protein expression, in particular, types, I, IV andVI collagen expression, and elastin and fibronectin expression. Inaddition, the influence of the extract on skin melanin expression isalso of considerable interest.

The effect of the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure on expression of these andother important cutaneous proteins can be monitored by human genomicmicroarray analysis and protein expression as well as by non-invasivetest methods well-known to those in skilled in the art, including, butnot limited to, improved moisturization, wrinkle reduction, reducedpigmentation, improved skin tone and the like. Application of themetabolized conditioned growth media and/or metabolized cell extract ofthe disclosure can manifest itself by measured reductions in skinwrinkles, for example, as measured by techniques such as SiIFIo siliconemodeling, PRIMOS and VISIO photographic systems and the like. Inaddition, moisturization might be measured using transepidermal waterloss (TEWL) or cutometer or corneometric measurements. Likewise, skinpigmentation could be measured using a chromometer. Such testingtechnologies are well-known to those skilled in the art and can be foundin the “Handbook of Non-Invasive Methods and the Skin”, 2_(nd) edition,Serup J, Jemec G B E, Grove G L (ed.), Taylor and Francis Boca RatonFla. 2006.

Human gene microarray analysis on human epidermal keratinocytesindicates that the metabolized conditioned growth medium up-regulatesseveral genes involved in various skin functions. See Example 18. It isthus appreciated that the yeast-metabolized conditioned growth media ormetabolized cell extract according to the present disclosure can be usedin personal care compositions for the treatment of skin conditions.

B. Wound Healing Applications

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure may be processed to promote wound and burn healing.When tissue is injured, polypeptide growth factors, which exhibit anarray of biological activities, are released into the wound to promotehealing. Wound healing is a complex process that involves several stagesand is capable of sealing breaches to the integument in a controlledmanner to form functionally competent tissue. The process begins withhemostasis followed by an inflammatory phase involving neutrophils andmacrophages. The process continues with the development of granulationtissue and re-epithelialization to close the wound. Subsequently, scartissue forms and is remodeled over the succeeding months to anapproximation of the original anatomical structure. Ideally, scar tissueis minimal so that healthy tissue, functionally competent tissue whichhistologically and physiologically resembles the original normal tissue,may form.

Each stage of the healing process is controlled by cellular interactionsthrough regulatory proteins such as cytokines, growth factors, andinflammatory mediators as well as cell contact mechanisms. For example,inflammatory mediators such as IL-6, IL-8, and G-CSF induce lymphocytedifferentiation and acute phase proteins, as well as neutrophilinfiltration, maturation and activation, processes that are important inthe inflammatory stages of wound healing. Other examples of regulatoryproteins involved in the wound healing process are VEGF that inducesangiogenesis during inflammation and granulation tissue formation, theBMP's which induce bone formation, KGF that activates keratinocytes andTGF-β1 that induces deposition of extracellular matrix.

In chronic wounds, the healing process is interrupted at a pointsubsequent to hemostasis and prior to re-epithelialization, and isapparently unable to restart. Most of the inflammation seen in the woundbed is related to infection, but the inflammation gives rise to anenvironment rich in proteases that degrade regulatory proteins and thusinterfere with the wound healing process.

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure contains activity thought to be important in woundhealing and which have been shown to be depleted in in vivo models ofwound healing. For example, the metabolized conditioned growth mediaand/or metabolized cell extract of the disclosure may contain anactivity of one or more of a number of growth factors that supporthealing process, such as VEGF, G-CSF, IL-8, KGF, and TGF-β. Furthermore,in some medical conditions, such as diabetes, some of the regulatoryproteins needed for wound healing are in short supply. For example, ithas been found in a mouse model of non-insulin-dependent diabetes (e.g.,the db/db mouse) that secretion of VEGF and PDGF and expression of thePDGF receptor are all depressed in wounds compared to the levels inwounds of normal mice.

Also, the metabolized conditioned growth media and/or metabolized cellextract of the disclosure is useful in the treatment of other types oftissue damage, e.g., traumatic or congenital, wherein the repair and/orregeneration of tissue defects or damage is desired since an activity ofmany of these growth factors are found in Applicants' metabolizedconditioned growth media and/or metabolized cell extract, including, forexample, fibroblast growth factors (FGFs), platelet derived growthfactors (PDGFs), epidermal growth factors (EGFs), bone morphogeneticproteins (BMPs) and transforming growth factors (TGFs); as well as thosewhich modulate vascularization, such as vascular endothelial growthfactor (VEGF), keratinocyte growth factor (KGF), and basic FGF;angiogenesis factors, and antiangiogenesis factors. Stress proteins,such as GR 78 and MSP90 induce growth factors such as TGF-β. TGF-β,including TGF β-1, TGF β-2, TGF β-3, TGF β-4 and TGF β-5, regulategrowth and differentiation and accelerate wound healing (Noda et al.1989, Endocrin. 124: 2991-2995; Goey et al. 1989, J. Immunol. 143:877-880, Mutoe et al. 1987, Science 237: 1333-1335). Mitogens, such asPDGF increase the rate of cellularity and granulation in tissueformation (Kohler et al. 1974, Exp. Cell. Res. 87: 297-301). Aspreviously mentioned, the cells are preferably human to minimizeimmunogenicity problems.

Because the metabolized conditioned growth media and/or metabolized cellextract of the disclosure contains an activity of an array of woundhealing factors, the conditioned media is advantageously used in thetreatment of wound and burn healing including skin wounds, broken bones,gastric ulcers, pancreas, liver, kidney, spleen, blood vessel injuriesand other internal wounds. Further, the metabolized conditioned growthmedia and/or metabolized cell extract of the disclosure may be combinedwith other medicinal ingredients such as antibiotics and analgesics.Embodiments include formulations of the metabolized conditioned growthmedia and/or metabolized cell extract of the disclosure with a salve orointment for topical applications.

Alternatively, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may be combined with abandage (adhesive or non-adhesive) to promote and/or accelerate woundhealing.

Wounds at specialized tissues may require medium conditioned by thatspecialized tissue. For example, injuries to neuronal tissues mayrequire proteins contained in medium conditioned by neuronal cellcultures. Specific products may be derived, or alternatively, themetabolized conditioned growth media and/or metabolized cell extract maybe enriched by immunoaffinity chromatography or enhanced expression of adesired protein from the specific medium such as, for example, NGF.NGF-controlled features include, but are not limited to, the cholinergicneurotransmitter function (acetylcholinesterase (AChE) and theacetylcholine-synthesizing enzyme (ChAT)), neuronal cell size, andexpression of Type II NGF receptors; NGF is secreted into theconditioned medium conditioned by glial and other neuronal cellscultured on a three-dimensional stromal tissue, which can then be usedin a composition for nerve healing.

Deficits of endogenous NGF aggravate certain human neurodegenerativedisorders and there is an apparent inability of injured adult CNSneurons to regenerate. Specifically, injury to a nerve is followed bydegeneration of the nerve fibers distal to the injury, the result ofisolation of the axon from the cell body. In the central nervous system,there is no significant growth at the site of injury typically leadingto death of the damaged neuron. NGF plays a crucial role in theregenerative capabilities of adult CNS cholinergic neurons at the cellbody level (e.g., septum), the intervening tissue spaces (e.g., nervebridge) and the reinervation area (e.g., hippocampal formation).Additionally, NGF may be beneficial in improving cognitive defects.Metabolized conditioned growth media and/or metabolized cell extractconditioned with glial cells for example, can supply an activity ofexogenous NGF and other nerve growth factors so that new axons can growout from the cut ends of the injured nerve (e.g., develop a growth cone)elongating to the original site of the connection.

Further, injury to the brain and spinal cord is often accompanied by aglial response to the concomitant axonal degeneration, resulting in scartissue. This scar tissue was initially thought to be a physical barrierto nerve growth, however, of greater significance is the presence orabsence of neuronotropic factors in the extra neuronal environment.Astrocytes appear to be capable of synthesizing laminin in response toinjury (laminin can also be found in the metabolized conditioned growthmedia). Collagen and fibronectin, and especially laminin, have beenfound to promote the growth of neurities from cultured neurons orneuronal explants in vitro. These extracellular matrix proteins appearto provide an adhesive substratum which facilitates the forward movementof the growth cone and elongation of the axon. Thus, the presence ofneuronotropic factors and a supportive substratum are required forsuccessful nerve regeneration since regeneration appears to requirethat: the neuronal cell body be capable of mounting the appropriatebiosynthetic response; and the environment surrounding the injury sitebe capable of supporting the elongation and eventual functionalreconnection of the axon. Metabolized conditioned growth media and/ormetabolized cell extract conditioned by nerve cells such as astrocytesand glial cells contains an activity of neuronotropic growth factors andextracellular matrix proteins necessary for nerve regeneration in brainand spinal cord injuries. Thus, in one embodiment, the metabolizedconditioned growth media and/or metabolized cell extract is formulatedfor the treatment of such injuries.

In other embodiments, the treatment of skin, bones, liver, pancreas,cartilage, and other specialized tissues may be treated with mediaconditioned by their respective specialized cell types, preferablycultured in three-dimensions, resulting in a conditioned mediumcontaining an activity of one or more characteristic extracellularproteins and other metabolites of that tissue type useful for treatingwounds to that respective tissue type.

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure may also be added to devices used in periodontalsurgery in order to promote uniform tissue repair, to providebiodegradable contact lenses, corneal shields or bone grafts, to providesurgical space fillers, to promote soft tissue augmentation,particularly in the skin for the purpose of reducing skin wrinkles, andas urinary sphincter augmentation, for the purpose of controllingincontinence.

In another embodiment, the compositions may be lyophilized/freeze-driedand added as a wound filler (e.g., fill holes left from hair plugs forimplantation) or added to existing wound filling compositions toaccelerate wound healing. In another embodiment, the medium isconditioned with genetically engineered cells to increase theconcentration of wound healing proteins in the medium. For example, thecells may be engineered to express gene products such as any of thegrowth factors listed above.

C. The Repair and Correction of Congenital Anomalies, Acquired Defectsand Cosmetic Defects

As disclosed above, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may also be used to repairand correct a variety of anomalies, both congenital and acquired as wellas cosmetic defects, both superficial and invasive. In one embodimentthe metabolized conditioned growth media and/or metabolized cell extractof the disclosure may be used to treat or prevent fine lines andwrinkles; age spots and dyspigmentation; decreased skin texture, toneand elasticity; roughness and photo damage; decreased ability of skin toregenerate itself; environmental damage; decreased smoothness andtightness of skin; age spots; fine and coarse lines and wrinkles; fineand coarse periocular wrinkles; nasolabial folds; facial fine and coarselines; decreased skin radiance and evenness; decreased skin firmness;hyperpigmentation; dark spots and/or patches; decreased skin brightnessand youthful appearance; photoaged skin; intrinsically and extrinsicallyaged skin; abnormal skin cellular turnover; decreased skin barrier;decrease of skin's ability to retain moisture; brown and redblotchiness; redness; abnormal skin epidermal thickness; reduction ofdermal epidermal junction; increased pore size and number of pores; or acombination thereof. In another embodiment, the metabolized conditionedgrowth media and/or metabolized cell extract of the disclosure may beused to rejuvenate sun damaged and aging skin; improve the appearance offine lines and wrinkles; promote cell renewal; and/or improve skin tone,texture and/or firmness. In another embodiment, the metabolizedconditioned growth media and/or metabolized cell extract of thedisclosure may be used to plump the skin and/or brighten and/or lightenthe skin. In another embodiment, the metabolized conditioned growthmedia and/or metabolized cell extract of the disclosure may be used tostrengthen the skin's ability to regenerate itself; improve theappearance of age spots; and/or improve skin firmness, elasticity,and/or resiliency. In another embodiment, the metabolized conditionedgrowth media and/or metabolized cell extract of the disclosure may beused to smooth, tighten, and/or fill in fine lines on the skin. Inanother embodiment, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may be used to reduce theappearance of dark circles under the eye. In another embodiment, themetabolized conditioned growth media and/or metabolized cell extract ofthe disclosure may be used to improve lip texture and/or condition,enhance natural lip color, and/or increase lip volume. In anotherembodiment, the metabolized conditioned growth media and/or metabolizedcell extract of the disclosure may be used to promote epithelializationof post-procedure skin and/or restore the skin's barrier and/or moisturebalance. In another embodiment, the metabolized conditioned growth mediaand/or metabolized cell extract of the disclosure may be used to improvethe appearance of age spots and/or pigmentation.

For example, compositions comprising metabolized conditioned growthmedia and/or metabolized cell extract of the disclosure may be added inany form and may be used in a hydrogel, injectable, cream, ointment, andmay even be added to eye shadow, pancake makeup, compacts or othercosmetics to fortify the skin topically.

In another embodiment, topical or application by any known method suchas injection, oral, etc., of the metabolized conditioned growth mediumis made to reverse and/or prevent wrinkles and a number of thedeleterious effects induced by UV light, exposure to a variety ofpollutants and normal aging for example.

Additionally, in another embodiment, the metabolized conditioned growthmedia and/or metabolized cell extract of the disclosure is used toreduce cell aging and inhibit the activity of the factors which causeskin cancer. That the metabolized conditioned growth media and/ormetabolized cell extract has antioxidant activity is shown in theexamples. Again, application to a mammal may be topical or applicationby any known method such as injection, oral, etc. Applicants havediscovered that a statistically significant (p<0.003) reduction inintracellular oxidation of approximately 50 percent was noted in humankeratinocytes exposed to Applicants' metabolized conditioned growthmedium.

Thus, in addition to inducing epidermal and dermal cell proliferationand collagen secretion in vitro the metabolized conditioned growth mediaand/or metabolized cell extract of the disclosure has antioxidantactivity. See Example 18.

This sterile enriched nutrient solution represents a bioengineeredcosmeceutical that is readily available in large volumes and may beuseful as an additive for a variety of skin, cosmetic, and dermatologicproducts to supplement the levels of growth factors and matrix moleculesin human skin, hair, and nails. Products are envisioned to use withAlpha Hydroxy Acids exfoliates to potentially optimize penetration ofthe growth factors and other biomolecules into the skin and withchemical peels to potentially accelerate healing and reduceinflammation.

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure may be formulated for eliminating wrinkles, frownlines, scarring and other skin conditions instead of using silicone orother products to do so. The metabolized conditioned growth media and/ormetabolized cell extract contains an activity of growth factors andinflammatory mediators such as, for example, VEGF, HGF, IL-6, IL-8,G-CSF and TFGβ, as well as extracellular matrix proteins such as type Iand type III collagens, fibronectin, tenascin, glycosaminologycans, acidand basic FGF, TGF-α and TGF-β, KGF, versican, decorin betaglycens,syndean and various other secreted human dermal matrix proteins whichare useful in repairing physical anomalies and cosmetic defects.

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure can be formulated into injectable preparations.Alternatively, products derived from the conditioned media can beformulated. For example, biologically active substances, such asproteins and drugs, can be incorporated in the compositions of thepresent disclosure for release or controlled release of these activesubstances after injection of the composition. Exemplary biologicallyactive substances can include tissue growth factors, such as TGF-β, andthe like which promote healing and tissue repair at the site of theinjection. Methods of product purification include, but are not limitedto gel chromatography using matrices such as SEPHADEX®, ion exchange,metal chelate affinity chromatography, with an insoluble matrix such ascross-linked agarose, HPLC purification, hydrophobic interactionchromatography of the conditioned media. Such techniques are describedin greater detail in Cell & Tissue Culture; Laboratory Procedures,supra; Sanbrook et al., 1989, Molecular Cloning: A Laboratory Manual,2^(nd) Ed., Cold Spring Harbor Lab Press, Cold Spring Harbor, N.Y.

In the injectable embodiment, an aqueous suspension is used and theformulation of the aqueous suspension may have a physiological pH (i.e.,about pH 6.8 to 7.5). Additionally, a local anesthetic, such aslidocaine, (usually at a concentration of about 0.3% by weight) isusually added to reduce local pain upon injection. The final formulationmay also contain a fluid lubricant, such as maltose, which must betolerated by the body. Exemplary lubricant components include glycerol,glycogen, maltose and the like. Organic polymer base materials, such aspolyethylene glycol and hyaluronic acid as well as non-fibrillarcollagen, preferably succinylated collagen, can also act as lubricants.Such lubricants are generally used to improve the injectability,intrudability and dispersion of the injected biomaterial at the site ofinjection and to decrease the amount of spiking by modifying theviscosity of the compositions. The final formulation is by definitionthe processed metabolized conditioned growth cell media in apharmaceutically acceptable carrier.

The processed metabolized conditioned growth media and/or metabolizedcell extract of the disclosure is subsequently placed in a syringe orother injection apparatus for precise placement of the metabolizedconditioned growth media and/or metabolized cell extract of thedisclosure at the site of the tissue defect. In the case of formulationsfor dermal augmentation, the term “injectable” means the formulation canbe dispensed from syringes having a gauge as low as 25 under normalconditions under normal pressure without substantial spiking Spiking cancause the composition to ooze from the syringe rather than be injectedinto the tissue. For this precise placement, needles as fine as 27gauges (200 μI.D.) or even 30 gauges (150 μI.D.) are desirable. Themaximum particle size that can be extruded through such needles will bea complex function of at least the following: particle maximumdimension, particle aspect ratio (length:width), particle rigidity,surface roughness of particles and related factors affectingparticle:particle adhesion, the viscoelastic properties of thesuspending fluid, and the rate of flow through the needle. Rigidspherical beads suspended in a Newtonian fluid represent the simplestcase, while fibrous or branched particles in a viscoelastic fluid arelikely to be more complex.

The above described steps in the process for preparing injectablesecreted metabolized conditioned growth media and/or metabolized cellextract of the disclosure are preferably carried out under sterileconditions using sterile materials. The processed metabolizedconditioned growth media and/or metabolized cell extract of thedisclosure in a pharmaceutically acceptable carrier can be injectedintradermally or subcutaneously to augment soft tissue, to repair orcorrect congenital anomalies, acquired defects or cosmetic defects.Examples of such conditions are congenital anomalies as hemifacialmicrosomia, malar and zygomatic hypoplasia, unilateral mammaryhypoplasia, pectus excavatum, pectoralis agenesis (Poland's anomaly) andvelopharyngeal incompetence secondary to cleft palate repair orsubmucous cleft palate (as a retropharyngeal implant); acquired defects(post-traumatic, post-surgical, post-infectious) such as depressedscars, subcutaneous atrophy (e.g., secondary to discoid lupiserythematosus), keratotic lesions, enophthalmos in the unucleated eye(also superior sulcus syndrome), acne pitting of the face, linearscleroderma with subcutaneous atrophy, saddle-nose deformity, Romberg'sdisease and unilateral vocal cord paralysis; and cosmetic defects suchas glabellar frown lines, deep nasolabial creases, circum-oralgeographical wrinkles, sunken cheeks and mammary hypoplasia. Thecompositions of the present disclosure can also be injected intointernal tissues, such as the tissues defining body sphincters toaugment such tissues.

Other tissue types used to condition the media include but are notlimited to bone marrow, skin, epithelial cells, and cartilage, however,it is expressly understood that the three-dimensional culture system canbe used with other types of cells and tissues.

Alternatively, the metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may be formulated withpolymerizable or cross-linking hydrogels as described in the previoussection on wound treatment.

D. Pharmaceutical Applications

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure contain a variety of useful pharmaceutical factors andcomponents such as growth factors, regulatory factors, peptide hormones,antibodies, etc., as described throughout the specification and aretherefore useful for a variety of pharmaceutical applications. Also,products which may be added include, but are not limited to,antibiotics, antivirals, antifungals, steroids, analgesics, antitumordrugs, investigational drugs or any compounds which would result in acomplimentary or synergistic combination with the factors in theconditioned media. As previously discussed, the cells are cultured, andthe media recovered under aseptic conditions. Additionally, the mediacan be tested for pathogens. If sterilization is done, it must be donein a manner which minimally affects the desired biological activity asdescribed, supra. The medium may be further processed to concentrate orreduce one or more factor or component contained within the medium, forexample, enrichment of a growth factor using immunoaffinitychromatography or, conversely, removal of a less desirable component,for any given application as described therein. In a preferredembodiment, formulations are made from medium conditioned by athree-dimensional cell construct. The three-dimensional cultures producea multitude of growth factors and proteins that are secreted into themedium at optimal physiological ratios and concentrations. See forexample, Table 2. The medium, therefore, provides a unique combinationof factors and specified ratios that closely represent those found invivo. Bovine serum is generally not preferred in this application. Itmay be preferable to remove cellular debris or other particular matteras well as proteases, lactic acid and other components possiblydetrimental to cell growth.

Assays commonly employed by those of skill in the art may be utilized totest the activity of the particular factor or factors, thereby ensuringthat an acceptable level of biological activity (e.g., a therapeuticallyeffective activity) is retained by the attached molecule or encapsulatedmolecule.

Thus, the metabolized conditioned growth media and/or metabolized cellextract of the disclosure and products derived from the media may beused, for example, to provide insulin in the treatment of diabetes,nerve growth factor for the treatment of Alzheimer's disease, factorVIII and other clotting factors for the treatment of hemophilia,dopamine for the treatment of Parkinson's disease, enkaphalins viaadrenal chromaffin cells for the treatment of chronic pain, dystrophinfor the treatment of muscular dystrophy, and human growth hormone forthe treatment of abnormal growth.

Doses of such therapeutic protein agents are well known to those ofskill in the art and may be found in pharmaceutical compedia such as thePHYSICIANS DESK REFERENCE, Medical Economics Data Publishers;REMINGTON′S PHARMACEUTICAL SCIENCES, Mack Publishing Co.; GOODMAN &GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw Hill Publ.,THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers.

The therapeutically effective doses of any of the drugs or agentsdescribed above may routinely be determined using techniques well knownto those of skill in the art. A “therapeutically effective” dose refersto that amount of the compound sufficient to result in amelioration ofat least one symptom of the processes and/or diseases being treated.

Toxicity and therapeutic efficacy of the drugs can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Additionally, the cells and tissues may be genetically engineered toenhance expression of a desired product such as insulin, for example,and/or to express nucleotide sequences and/or moieties which target thegene products listed above e.g. ribozyme, antisense molecules and triplehelices, which may have an inhibitory effect on target gene expressionand/or activity. This might be advantageous when culturing tissues inwhich specialized stromal cells in the medium may play particularstructural/functional roles, e.g., glial cells of neurological tissue,Kupffer cells of liver, etc.

E. Stimulation of Hair Growth

The medium may be conditioned using, for example, human hair papillacells. Preferably, the medium conditioned by such cells is grown inthree-dimensions. Hair papilla cells are a type of mesenchymal stem cellthat plays a pivotal role in hair formation, growth and restoration(Matsuzaki et al., Wound Repair Regen, 6:524-530 (1998)). Thesubsequently metabolized conditioned growth medium is preferablyconcentrated and applied as a topical formulation. The metabolizedconditioned media compositions may be formulated for topicalapplications using an agent that facilitates penetration of the compoundinto the skin, for example, DMSO, and applied as a topical applicationfor stimulating hair growth.

The compositions of the disclosure promote or restore hair growth whenapplied topically by providing factors that increase epithelial cellmigration to hair follicles. In addition to the factors found in themetabolized conditioned media, other compounds, such as minoxidil andantibiotics can be used. During hair growth there is a reduction inblood supply during catagen (the transitional phase of the hair folliclebetween growth and resting phases) and telogen (the resting phase).Biologically active molecules derived from the metabolized conditionedcell medium can be determined and optimized for use during these phasesof hair growth using assays known in the art including the stump-tailedmacaque model for male-patterned baldness, see for example, Brigham,Pa., A. Cappas, and H. Uno, The Stumptailed Macaque as a Model forAndrogenetic Alopecia: Effects of Topical Minoxidil Analyzed by Use ofthe Folliculogram, Clin Dermatol, 1988, 6(4): p. 177-87; Diani, A. R.and C. J. Mills, Immunocytochemical Localization of Androgen Receptorsin the Scalp of the Stumptail Macaque Monkey, a Model of AndrogeneticAlopecia, J Invest Dermatol, 1994, 102(4): p. 511-4; Holland, J. M.,Animal Models of Alopecia, Clin Dermatol, 1988, 6(4): p. 159-162; Pan,H. J., et al., Evaluation of RU58841 as an Anti-Androgen in Prostate PC3Cells and a Topical Anti-Alopecia Agent in the Bald Scalp of StumptailedMacaques, Endocrine, 1998, 9(1): p. 39-43; Rittmaster, R. S., et al.,The Effects of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17beta-carboxamide, a 5 alpha-reductase Inhibitor and Antiandrogen, on theDevelopment of Baldness in the Stumptail Macaque, J. Clin EndocrinolMetab, 1987, 65(1): p. 188-93 (each of which is incorporated byreference in its entirety). Additional models include measuringdifferences in hair follicle proliferation from follicles cultured frombald and hairy areas, a newborn rat model as well as a rat model ofalopecia greata, see, Neste, D. V., The Growth of Human hair in NudeMice, Dermatol Clin., 1996, 14(4): p. 609-17; McElwee, K. J., E. M.Spiers, and R. F. Oliver, In Vivo Depletion of CD8+T Cells Restores HairGrowth in the DEBR Model for Alopecia Areata, Br J Dermatol, 1996,135(2): p. 211-7; Hussein, A. M., Protection Against CytosineArabinowide-Induced Alopecia by Minoxidil in a Rat Animal Model, Int JDermatol, 1995, 34(7): p. 470-3; Oliver, R. F., et al., The DEBR RatModel for Alopecia Areata, J Invest Dermatol, 1991, 96(5): p. 978;Michie, H. J., et al., Immunobiological Studies on the Alopecic (DEBER)Rat, Br J Dermatol, 1990, 123(5): p. 557-67 (each of which isincorporated by reference in its entirety).

V. OTHER USES

A. Food Additives and Dietary Supplements

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure may be used as food additives and formulated intodietary supplements. The metabolized conditioned growth media and/ormetabolized cell extract of the disclosure may contain many usefulnutrients including essential amino acids, minerals, and vitamins in anabundance and variety not found in individual foods or good groups. Themetabolized conditioned growth media and/or metabolized cell extract ofthe disclosure can be used as an inexpensive source for a balancednutritional supplement for weight loss or alternatively for enhancingthe nutritional content of food, particularly for third world countries.The metabolized conditioned growth media and/or metabolized cell extractof the disclosure is sterile and is free from contamination by humanpathogens (i.e., aseptic). The metabolized conditioned growth mediaand/or metabolized cell extract of the disclosure may be concentratedand/or lyophilized and preferably administered in capsules or tabletsfor ingestion. Alternatively, the compositions may be directly added toadult or baby food to enhance nutritional content. This rich source ofnutrients may be processed relatively inexpensively and can beinvaluable to undernourished elderly people, and in particular, tochildren in underdeveloped countries where increased mortality due topoor responses to infection have been associated with malnutrition.

Additionally, many trace elements, such as iron and magnesium, arecritical for mammalian survival and reproduction, and there is concernthat marginal trace element deficiency may be a public health problem.The intake of various essential micronutrients has been suggested todecrease infection as well as cancer risk by modifying specific phasesof carcinogenesis. Micronutrients also enhance the functional activitiesof the immune system and its interacting mechanism of T cells and Bcells, Mos, and NK cells specifically by enhancing the production ofvarious cytokines to facilitate their phagocytic and cytotoxic actionagainst invading pathogens and/or to destroy emerging premalignant cellsin various vital organs. See, Chandra, R. K. ed. (1988), Nutrition andImmunology. Contemporary Issues in Clinical Nutrition, Alan R. Liss, NewYork. Thus, there is a need for a relatively inexpensive source ofbalanced nutrients. Ideal food products for enrichment with theconditioned media are breads, cereals and other grain products such aspastas, crackers, etc. Also, the metabolized medium may be furtherprocessed to concentrate or reduce one or more factor or componentcontained within the medium, for example, enrichment of a growth factorusing immunoaffinity chromatography or, conversely, removal of a lessdesirable component, for any given application as described in thissection.

B. Animal Feed Supplement

The metabolized conditioned growth media and/or metabolized cell extractof the disclosure may be used as a supplemental to animal feed. In oneembodiment, the metabolized conditioned growth media and/or metabolizedcell extract of the disclosure contains bovine serum that provides asource of protein and other factors that are beneficial for mammals suchas cattle and other ruminant animals, such as cows, deer and the like.The medium is screened for pathogens and is free of bovine pathogens andmycoplasma. The metabolized conditioned growth media and/or metabolizedcell extract of the disclosure is preferably obtained from cows raisedin the United States so that the likelihood of pathogens is markedlydiminished.

In one embodiment, provided are compositions comprising a metabolizedconditioned growth medium and an acceptable carrier, wherein themetabolized conditioned growth medium is conditioned growth mediummetabolized by yeast cells. In some embodiments, the conditioned growthmedium is prepared by culturing cells in a growth medium sufficient tomeet the nutritional needs required to grow the cells in vitro to form aconditioned growth medium. In some embodiments, the composition is aninjectable composition or a topical composition. In some embodiments,the topical composition is an ointment, a cream, a hydrogel, or alotion. In other embodiments, the composition is used to treat orprevent a cosmetic defect, a congenital defect, hair loss or an aquireddefect. In other embodiments, the cosmetic defect is a glabellar frownline, deep nasolabial crease, circum-oral geographical wrinkle, sunkencheeks or mammary hypoplasia. In some embodiments, the acquired defectis a medical condition that occurs post-trauma, post-surgery,post-infection or post-medical procedure defect.

In other embodiments, provided are compositions comprising a metabolizedcell extract and an acceptable carrier, wherein the metabolized cellextract is cell extract metabolized by yeast cells. In some embodiments,the cell extract is derived from fibroblasts. In other embodiments, thecomposition is an injectable composition or a topical composition. Inyet other embodiments, the topical composition is an ointment, a cream,a hydrogel, or a lotion. In still other embodiments, the composition isused to treat or prevent a cosmetic defect, a congenital defect, hairloss or an aquired defect. In some embodiments, the cosmetic defect is aglabellar frown line, deep nasolabial crease, circum-oral geographicalwrinkle, sunken cheeks or mammary hypoplasia. In yet other embodiments,the acquired defect is a medical condition that occurs post-trauma,post-surgery, post-infection or post-medical procedure defect.

In still other embodiments, provide are metabolized conditioned growthmedium prepared by a process comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium.

In some embodiments, the yeast is Pichia pastoris. In other embodiments,the cells in step (a) are fibroblasts. In yet other embodiments, theprocess further comprises (e) processing the metabolized conditionedgrowth medium, wherein processing is concentrating, filtering,purifying, or a combination thereof.

In yet other embodiments, provided are processes for preparing themetabolized conditioned growth medium of any one of claims 15 to 18,comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium.

In some embodiments, the metabolized conditioned growth medium iscombined with an acceptable carrier. In yet other embodiments, themetabolized conditioned growth medium is present in an amount of about0.0001% to about 95% by weight of the composition, about 0.01% to about50% by weight of composition or about 0.01% to about 10% by weight ofcomposition. In still other embodiments, the composition furthercomprises at least one of water, surfactants, emulsifiers, conditioners,emollients, waxes, oils, polymers, thickeners, fixatives, colorants,nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,antifungals, antimicrobials, steroidal hormones, antidandruff agents,anti-acne components, sunscreens, and preservatives. In still otherembodiments, the composition is an injectable composition or a topicalcomposition. In some embodiments, the topical composition is anointment, a cream, a hydrogel, or a lotion.

Also provided herein are metabolized cell extract prepared by a processcomprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract.

In some embodiments, the yeast is Pichia pastoris. In other embodiments,the cell extract is derived from fibroblasts. In some embodiments, theprocess further comprises (e) processing the metabolized cell extract,wherein processing is concentrating, filtering, purifying, or acombination thereof.

In one embodiment, provided are compositions comprising: an acceptablecarrier and a metabolized conditioned growth medium prepared by aprocess comprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium.

In some embodiments, the yeast is Pichia pastoris. In other embodiments,the cells in step (a) are fibroblasts. In some embodiments, thecomposition further comprises (e) processing the metabolized conditionedgrowth medium, wherein processing is concentrating, filtering,purifying, or a combination thereof. In some embodiments, themetabolized conditioned growth medium is present in an amount of about0.0001% to about 95% by weight of the composition, about 0.01% to about50% by weight of the composition, or about 0.01% to about 10% by weightof the composition. In other embodiments, the composition furthercomprises at least one of water, surfactants, emulsifiers, conditioners,emollients, waxes, oils, polymers, thickeners, fixatives, colorants,nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,anifungals, antimicrobials, steroidal hormones, antidandruff agents,anti-acne components, sunscreens, and preservatives. In someembodiments, the composition is an injectable composition or a topicalcomposition. In yet other embodiments, the topical composition is anointment, a cream, a hydrogel, or a lotion.

Also provided herein are compositions comprising: an acceptable carrierand a metabolized cell extract prepared by a process comprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract.

In one embodiment, the yeast is Pichia pastoris. In some embodiments,the cell extract is derived from fibroblasts. In other embodiments, theprocess further comprises (e) processing the metabolized cell extract,wherein processing is concentrating, filtering, purifying, or acombination thereof. In some embodiments, the metabolized conditionedgrowth medium is present in an amount of about 0.0001% to about 95% byweight of the composition, about 0.01% to about 50% by weight of thecomposition, or about 0.01% to about 10% by weight of the composition.In some embodiments, the composition further comprises at least one ofwater, surfactants, emulsifiers, conditioners, emollients, waxes, oils,polymers, thickeners, fixatives, colorants, nutraceuticals,cosmeceuticals, therapeutics, pharmaceuticals, anifungals,antimicrobials, steroidal hormones, antidandruff agents, anti-acnecomponents, sunscreens, and preservatives. In other embodiments, thecomposition is an injectable composition or a topical composition. Instill other emboidments, the topical composition is an ointment, acream, a hydrogel, or a lotion.

Also provided herein are methods for preventing or treating a conditionin a subject comprising administering to the subject a therapeuticallyeffective amount of a composition comprising: an acceptable carrier anda metabolized conditioned growth medium prepared by a processcomprising:

(a) culturing cells in a growth medium sufficient to meet thenutritional needs required to grow the cells in vitro to form aconditioned growth medium and removing the conditioned growth mediumfrom the cultured cells;

(b) culturing yeast cells;

(c) exposing the yeast cells to the conditioned growth medium;

(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium; and

(e) collecting the metabolized conditioned growth medium.

In one embodiment, the condition is a cosmetic defect, hair loss, acongenital defect or an acquired defect. In other emboidments, theacquired defect is a medical condition that occurs post-trauma,post-surgery, post-infection or a post-medical procedure defect. In yetother embodiments, the cosmetic defect is a glabellar frown line, deepnasolabial crease, circum-oral geographical wrinkle, sunken cheeks ormammary hypoplasia. In still other embodiments, the yeast is Pichiapastoris. In some embodiments, the cells in step (a) are fibroblasts. Inother embodiments, the process further comprises (e) processing themetabolized conditioned growth medium, wherein processing isconcentrating, filtering, purifying, or a combination thereof. In someembodiments, the metabolized conditioned growth medium is present in thecomposition in an amount of about 0.0001% to about 95% by weight of thecomposition, about 0.01% to about 50% by weight of the composition, orabout 0.01% to about 10% by weight of the composition. In onenon-limiting example, the metabolized conditioned growth medium ispresent in the composition in an amount of about 10%, about 15%, orabout 20% by weight of the composition. In still other embodiments, thecomposition further comprises at least one of water, surfactants,emulsifiers, conditioners, emollients, waxes, oils, polymers,thickeners, fixatives, colorants, nutraceuticals, cosmeceuticals,therapeutics, pharmaceuticals, anifungals, antimicrobials, steroidalhormones, antidandruff agents, anti-acne components, sunscreens, andpreservatives. In yet other embodiments, the composition is aninjectable composition or a topical composition. In other embodiments,the topical composition is an ointment, a cream, a hydrogel, or alotion. In still other embodiments, at least one extracellular matrixprotein is up-regulated by the administration of the composition to thesubject. In one embodiment, the extracellular matrix protein isup-regulated by about 5% to about 100%, by about 10% to about 50%, or byabout 60% to about 100%. In another embodiment, the extracellular matrixprotein is a collagen protein or a lysyl hydroxylase protein. In stillanother embodiment, the extracellular matrix protein is encoded byCOL4A1 gene or PLOD1 gene. In one embodiment, at least one repairprotein is up-regulated by the administration of the composition to thesubject. In another embodiment, the repair protein is up-regulated byabout 10% to about 70%, by about 35% to about 50%, or by about 25% toabout 50%. In another embodiment, the repair protein is a fibronectinprotein. In another emboidment, the repair protein is encoded by FN1gene. In still another embodiment, at least one cellular connectivityprotein is up-regulated by the administration of the composition to thesubject. In some embodiments, the cellular connectivity protein isup-regulated by about 5% to about 200%, by about 10% to about 80%, or byabout 30% to about 50%. In some embodiments, the cellular connectivityprotein is involucrin protein. In still other embodiments, the cellularconnectivity protein is encoded by IVL gene. In one embodiment, at leastone antioxidant protein is up-regulated by the administration of thecomposition to the subject. In another embodiment, the antioxidantprotein is up-regulated by about 5% to about 300%, by about 10% to about50%, or by about 200% to about 250%. In another embodiment, theantioxidant protein is a superoxide dismutase protein. In still otherembodiments, the antioxidant protein is encoded by SOD2 gene.

Also provided herein are methods for preventing or treating a conditionin a subject comprising administering to the subject a therapeuticallyeffective amount of a composition comprising: an acceptable carrier anda metabolized cell extract prepared by a process comprising:

(a) providing a cell extract;

(b) culturing yeast cells;

(c) exposing the yeast cells to the cell extract;

(d) culturing the yeast cells to metabolize at least a portion of thecell extract; and

(e) collecting the metabolized cell extract.

In some embodiments, the condition is a cosmetic defect, hair loss, acongenital defect or an acquired defect. In some embodiments, theacquired defect is a medical condition that occurs post-trauma,post-surgery, post-infection, or post-medical procedure defect. In otherembodiments, the cosmetic defect is a glabellar frown line, deepnasolabial crease, circum-oral geographical wrinkle, sunken cheeks ormammary hypoplasia. In one embodiment, the yeast is Pichia pastoris. Inanother embodiment, the cell extract is derived from fibroblasts. Instill another embodiment, the process further comprises (e) processingthe metabolized cell extract, wherein processing is concentrating,filtering, purifying, or a combination thereof. In still otherembodiments, the metabolized conditioned growth medium is present in thecomposition in an amount of about 0.0001% to about 95% by weight of thecomposition, by about 0.01% to about 50% by weight of the composition,or by about 0.01% to about 10% by weight of the composition. In onenon-limiting example, the metabolized cell extract is present in thecomposition in an amount of about 10%, about 15%, or about 20% by weightof the composition. In still other embodiments, the composition furthercomprises at least one of water, surfactants, emulsifiers, conditioners,emollients, waxes, oils, polymers, thickeners, fixatives, colorants,nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,anifungals, antimicrobials, steroidal hormones, antidandruff agents,anti-acne components, sunscreens, and preservatives. In one embodiment,the composition is an injectable composition or a topical composition.In another embodiment, the topical composition is an ointment, a cream,a hydrogel, or a lotion. In one embodiment, at least one extracellularmatrix protein is up-regulated by the administration of the compositionto the subject. In another embodiment, the extracellular matrix proteinis up-regulated by about 5% to about 100%, by about 10% to about 50%, orby about 60% to about 100%. In yet another embodiment, the extracellularmatrix protein is a collagen protein or a lysyl hydroxylase protein. Insome embodiments, the extracellular matrix protein is encoded by COL4A1gene or PLOD1 gene. In some embodiments, at least one repair protein isup-regulated by the administration of the composition to the subject. Inone embodiment, the repair protein is up-regulated by about 10% to about70%, or by about 25% to about 50%. In another embodiment, the repairprotein is a fibronectin protein. In still another embodiment, therepair protein is encoded by FN1 gene. In one embodiment, at least onecellular connectivity protein is up-regulated by the administration ofthe composition to the subject. In another embodiment, the cellularconnectivity protein is up-regulated by about 5% to about 200%, by about10% to about 80%, or by about 30% to about 50%. In still anotherembodiment, the cellular connectivity protein is involucrin protein. Instill another embodiment, the cellular connectivity protein is encodedby IVL gene. In one embodiment, at least one antioxidant protein isup-regulated by the administration of the composition to the subject. Inanother embodiment, the antioxidant protein is up-regulated by about 5%to about 300%, by about 10% to about 50%, or by about 200% to about250%. In another embodiment, the antioxidant protein is a superoxidedismutase protein. In yet another embodiment, the antioxidant protein isencoded by SOD2 gene.

EXAMPLES Example 1 Fibroblast Monolayer Cell Culture

Normal human dermal fibroblasts, isolated from a human foreskin, werecultured in a 150 cm² tissue culture flasks (Corning, Corning, N.Y.) inmonolayer culture using pre-conditioned cell culture media (in thisexample, high-glucose Dulbecco's Modified Eagle's Media (DMEM; GibcoBRL,Grand Island, N.Y.) supplemented with 10% bovine calf serum (HycloneLaboratories, Logan, Utah), nonessential amino acids (GibcoBRL), and 100U/ml penecillin-streptomycin-250 ng/ml amphoterecin B (GibcoBRL) (“DMEM1”) in a 37° C., 5% CO₂ incubator. Monolayer cultures were fed twiceweekly with fresh pre-conditioned media and passaged weekly using a 1 to10 split, as described. See generally, Pinney et al., J. Cell. Physio.,183:74 82 (2000). The dermal fibroblasts may also be expanded in rollerbottles with DMEM 1. The conditioned media from these monolayer culturesis collected and saved for future use.

While fibroblast cells have been used for illustrative purposes in thisexample, the skilled artisan will understand that many other types ofcells, for example, but not limited to, other epithelial cell types,endothelial cells, smooth muscle cells, myocytes, keratinocytes,chondrocytes, and the like, may be grown in monolayer culture and inthree-dimensional culture.

Example 2 Conditioning the Medium

Human dermal fibroblasts were seeded onto the substrate of the apparatusdescribed in the '766 patent and described in detail above. Thesubstrate is within the casing designed to facilitate three-dimensionaltissue growth on its surface and the cells were cultured in a closedsystem in cultured in high glucose DMEM (10% BCS supplemented with 2 mML-glutamine and 50 mg/ml ascorbic acid) at 37° C. in a humidified, 5%CO₂ atmosphere. After 10 days the cell culture was removed, fresh mediumwas added. The cells were cultured for an additional 4 days as describedabove. The resulting conditioned medium, having been exposed to the celland tissue culture for four days (days 10-14) was then removed from theindividual chambers and pooled. The conditioned medium (approximately 5to 10 liters/pool) was dispensed into 200 ml aliquots and furtherconcentrated 10- to 20-fold using a positive pressure concentrationdevice having a filter with a 10,000 MW cut-off (Amicon, Beverly,Mass.). The resulting 10 to 20 ml of concentrated conditioned medium wasdispensed into 1 ml aliquots and frozen at −20° C. for analysis. A 1×concentration of conditioned medium results from 10× conditioned mediumadded to base medium as a 10% (vol/vol) solution. Likewise, a 1×concentration of “medium” or “serum free medium” results from 10× medium(i.e., base medium) or 10× serum free medium (base medium without serum)added to base medium as a 10% (vol/vol) solution which are then used ascontrols.

Example 3 Preparation of Metabolized Conditioned Growth Media

a. Organism and Media

Pichia pastoris (wild-type strain) was used as the yeast cell culturefor fermentation work. Stock cultures were maintained onyeast-peptone-dextrose (YPD) agar plates. The parent stock culture wasgrown in YPD broth and maintained at −20° C. The fermentation wascarried out in Yeast Nitrogen-Base (YNB) growth media and supplementedwith glycerol containing, 2.7% H₃PO₄, 0.09% CaSO₄, 1.8% K₂SO₄, 1.5%MgSO₄, 0.41% KOH, 4% glycerol (Sigma St. Louis, Mo.). Condition GrowthMedia was supplied by SkinMedica (California).

b. Fermentor

The fermentation process was scaled up to 2L and 15L fermentation stages(2L New Brunswick Scientific, Edison N.J. and 15L Applikon BiotechnologyFoster City Calif.) using standard growing conditions for the yeast.

c. Conditioned Growth Media Metabolization Conditions

Fed-batch cultures were grown at 29° C., in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol (2.7% H₃PO₄, 0.09% CaSO₄,1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol). The pH was keptconstant at 5.0±0.5 by titration with 2M NH₄OH. The dissolved oxygenlevels were measured by a sterilizable DO probe and the oxygensaturation was kept at 30% by regulating the stirring velocity between100 and 600 rpm. Conditioned growth media was introduced to thefermentation process as a nutritional supplement by inoculating theliquid media into the fermentor during the active growth phase of theyeast. The fed-batch process was initiated during the logarithmic growthphase of Pichia pastoris. Growth was measured by monitoring opticaldensity of Pichia pastoris during fermentation. The fermentation wascarried out until such time that the conditioned growth media wasmetabolized by Pichia pastoris, as measured by the changes in the growthphase of Pichia pastoris from logarithmic phase to stationary phase. Thefermentation may be carried out to completion wherein the conditionedgrowth media is fully metabolized, or may be partially fermented, i.e.,partially metabolized.

The conditioned growth media may be concentrated, filtered and/orpurified prior to combining the metabolized conditioned growth mediumwith a suitable vehicle.

Example 4 Preparation of Metabolized Cell Extract

a. Cell Extract

Whole cell extract is obtained from animal cells or plant cells, forexample, by any method known to the skilled artisan.

b. Organism and Media

Pichia pastoris (wild-type strain) is used as the yeast cell culture forfermentation work. Stock cultures are maintained onyeast-peptone-dextrose (YPD) agar plates. The parent stock culture isgrown in YPD broth and maintained at −20° C. The fermentation is carriedout in Yeast Nitrogen-Base (YNB) growth media and supplemented withglycerol containing, 2.7% H₃PO₄, 0.09% CaSO₄, 1.8% K₂SO₄, 1.5% MgSO₄,0.41% KOH, 4% glycerol (Sigma St. Louis, Mo.).

b. Fermentor

The fermentation process is scaled up to 2L and 15L fermentation stages(2L New Brunswick Scientific, Edison N.J. and 15L Applikon BiotechnologyFoster City Calif.) using standard growing conditions for the yeast.

c. Cell Extract Metabolization Conditions

Fed-batch cultures are grown at 29° C., in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol (2.7% H₃PO₄, 0.09% CaSO₄,1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol). The pH is kept constantat 5.0±0.5 by titration with 2M NH₄OH. The dissolved oxygen levels aremeasured by a sterilizable DO probe and the oxygen saturation was keptat 30% by regulating the stirring velocity between 100 and 600 rpm. Cellextract is introduced to the fermentation process as a nutritionalsupplement by inoculating the liquid media into the fermentor during theactive growth phase of the yeast. The fed-batch process is initiatedduring the logarithmic growth phase of Pichia pastoris. Growth ismeasured by monitoring optical density of Pichia pastoris duringfermentation. The fermentation is carried out until such time that thecell extract is metabolized by Pichia pastoris, as measured by thechanges in the growth phase of Pichia pastoris from logarithmic phase tostationary phase. The cell extract may be may be concentrated, filteredand/or purified prior to combining the metabolized cell extract with asuitable vehicle.

d. Proliferating Activity of Three-Dimensional-Conditioned Medium

The conditioned medium and metabolized conditioned growth medium andmetabolized cell extract of Examples 2-4 may be optionally examined forthe ability to promote the proliferation of human fibroblasts andkeratinocytes. Human fibroblasts or human basal keratinocytes are seededinto 96 well plates (˜5,000 cells/well) and cultured in high glucoseDMEM (10% BCS supplemented with 2 mM L-glutamine and 1×antibiotic/antimycotic) supplemented with 1× final concentration ofserum-free-medium, medium, or the three-dimensional conditioned medium,metabolized conditioned growth medium or metabolized cell extractasdescribed above in Examples 2-4. The cultures are maintained at 37° C.in a humidified, 5% CO₂ atmosphere for 3 days.

Cellular proliferation is measured using a commercially available,fluorescent-based dye assay that measures total nucleic acid content asan estimation of cell proliferation (CyQuant Cell Proliferation AssayKit, Molecular Probes, Eugene, Or). All assays are performed accordingto the manufacturer's instructions. Medium is removed by blotting andthe cells are lysed using lysis buffer containing the green fluorescentdye, CyQuant GR dye. The dye exhibits strong fluorescence enhancementwhen bound to cellular nucleic acids and the amount of fluorescence isproportional to the amount of nucleic acid present in the sample.Samples are incubated for 5 minutes in the absence of light and samplefluorescence is determined using a microtiter plate reader with filtersappropriate for ˜480 nm excitation and ˜520 nm emission maxima. Theamount of nucleic acid in each sample is calculated by comparing theamount of observed fluorescence in each well against a standard curve,derived using known concentrations of calf thymus DNA as a standard.

An exemplary result is shown in FIG. 3, where the cells cultured in themedium containing the conditioned medium resulted in increased cellularproliferation of both fibroblast and keratinocyte cells when compared totwo controls.

Example 5 Modulation of Collagen Deposition into Tissues by MetabolizedConditioned Growth Medium

a. Wound Healing Applications

The effect of metabolized conditioned growth medium on the preparationand composition of three-dimensional tissues is examined by measuringthe amount of collagen secreted into the extracellular matrix of tissuescultured in the presence of serum-free medium, medium or threedimensional conditioned medium.

Nylon scaffolds are laser-cut into 11 mm×11 mm squares, washed in 0.5Macetic acid, rinsed extensively in FBS, and seeded with 12F clinicalfibroblasts at passage 8 (˜38, 000/cm²). Cultures are grown in 1 ml ofDMEM (10% BCS supplemented with 2 mM L-glutamine and 1×antibiotic/antimycotic) supplemented with 1× final concentration ofserum-free-medium, medium, or the metabolized conditioned growth mediumas described above in with the addition of 50 mg/ml ascorbate at eachfeeding. Copper sulfate is added to a final concentration of 2.5 ng/ml,and high oxygen (40%, about twice atmospheric) is maintained byregulated gassing of a standard incubator. Cultures (n=3 or greater) aremaintained at 37° C. in a humidified, 5% CO₂ atmosphere for 10 days. Ano ascorbate control is also included.

b. Collagen Isolation

Collagen is isolated and purified to near homogeneity fromthree-dimensional tissue cultures grown in the presence of base mediumsupplemented with a 1× final concentration of serum-free medium, mediumor metabolized conditioned growth medium described above. The purity ofthe final samples preparations is estimated by subjecting the purifiedcollagen samples to electrophoresis on gradient SDS-polyacrylamide gels,visualizing the separate protein bands using Coomassie blue, andestimating the amount of collagen-specific alpha-, beta- and gamma-bandscompared to total protein (below). Purification methods should yieldsimilar patterns in all samples.

Samples are rinsed in PBS, then sterile water, followed by 2-6 hours in0.5M acetic acid. The samples are then digested overnight in 1 mg/mlpepsin (Worthington, Inc.) in 0.012N HCl at 4° C. Samples are clarifiedby centrifugation at 13000 rpm at 4° C. Collagen is precipitated 30-60minutes at 4° C. after addition of 5 M NaCl to a final concentration of0.7M. Precipitated collagen is separated by centrifugation at 13000 rpmat 4° C. for 30 to 60 minutes, and resuspended in 0.012N HCl.

c. Analysis

Total protein is determined using a commercially available colorimetricassay kit (Pierce, Inc. BCA assay kit) and assays are performedaccording to the manufacturer's instructions. Bovine skin collagens areused as a standard (InVitrogen, Carlsbad, Calif.; Cohesion Technologies,Inc., Palo Alto, Calif.) for quantifying total protein.

Samples (10 mg) are then subjected to SDS-PAGE analysis withelectrophoresis on 3-8% gradient gels. The samples of isolated collagensare then heated to 95° C. in reducing sample buffer. Gels are stainedwith Coomassie Blue, destained, and computer-scanned for visualization.

Enhanced deposition of collagen in vivo has a number of applications,including wound healing, the treatment of wrinkles and contour linesthat appear with increased age as well as being able to promote matrixdeposition over bony-prominences susceptible to pressure ulcers inparalyzed or bedridden patients.

Example 6 Occlusive Patch Test Assessing the In Vivo Effects ofMetabolized Conditioned Growth Medium

a. Experimental Design:

Six consenting adult females (30-60 yr) in good health are enrolled.Exclusion criteria includes sensitivity to proteins, skin diseases,damaged skin in or near test sites, diabetes, renal, heart orimmunological disorders, use of anti-inflammatory, immuno suppressive,antihistamine or topical drugs or cosmetics and pregnancy. Test articlesare assigned to test sites (2 sites, 3.8 cm²) on the right or leftforearm of each subject according to a rotational scheme to minimizeposition or order bias. Site 1 is designated for vehicle control andsite 2 treatment (i.e., metabolized conditioned growth medium).Occlusion patches are of a Webril nonwoven cotton pad with either 0.2 mlof vehicle or treatment. Patches are covered and held by a 3M occlusive,plastic, hypoallergenic tape. Occlusion patches are positioned daily onthe forearms of 3 subjects for 5 consecutive, 24-hour periods. Theremaining 3 subjects are patched daily for 12, consecutive 24-hourperiods. On the day following the last patch application, a 2-mm biopsyis taken from each site. This protocol is approved by the IRB for theinvestigative organization, the California Skin Research Institute (SanDiego, Calif.), and is in accordance with Title 21 of the CFR, Parts 50and 56.

b. Evaluations:

Gross observations are graded for glazing, peeling, scabbing, fissuring,hyperpigmentation, and hypopigmentation. Irritation is scored visuallyusing a 5 point scale and graded numerically for erythema, edema,papules and vesicles (>25% patch site), and identifiable reactions (<25%patch site), i.e., bulla reaction with or without weeping, spreading,and induration. The H & E histological assessment by a board certifiedpathologist includes parameters for viable epidermal thickness,epidermal hyperplasia (acanthosis), granular cell layer thickness,inflammatory infiltrate, mitotic figures, appearance of collagen andelastic fibers, and vasculature.

Example 7 Modulation of Human Endothelial Cell Behavior

The effects of metabolized conditioned growth medium on angiogenesis andendothelial cell motility are determined. Metabolized conditioned growthmedium is either concentrated (10×) or lyophilized.

a. Endothelial Cell Tubule Formation Assay

Endothelial cell tubule formation assay with human umbilical veinendothelial cells (HUVEC) is used to assess angiogenesis.

b. Wounding Assay

A confluent layer of endothelial cells are scratched and the speed ofclosure of the resulting “wound” is measured used to assess cellmotility. The “wounding” assay is measured as speed of closure in mm/h(millimeters/hour).

Example 8 Preparation of Yeast Extract

a. Organism and Media

Yeast cell culture was obtained from ATCC (Pichia pastoris #60372).Stock cultures were maintained on yeast-peptone-dextrose (YPD) agarplates. The parent stock culture was grown in YPD broth and maintainedat −20° C. The fermentation was carried out in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol containing, 2.7% H₃PO₄,0.09% CaSO₄, 1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol (Sigma St.Louis, Mo.). Antifoam sigma-emulsion B was used throughout the process(Sigma, St. Louis, Mo.).

b. Fermentor

After optimization of treatment via the shake flask trials, the processwas scaled up to 2L and 15L fermentation stages (2L New BrunswickScientific, Edison N.J. and 15L Applikon Biotechnology Foster CityCalif.).

c. Stress Conditions

Fed-batch cultures were grown at 29° C., in Yeast Nitrogen-Base (YNB)growth media and supplemented with glycerol (2.7% H₃PO₄, 0.09% CaSO₄,1.8% K₂SO₄, 1.5% MgSO₄, 0.41% KOH, 4% glycerol). The pH was keptconstant at 5.0±0.5 by titration with 2M NH₄OH. The dissolved oxygenlevels were measured by a sterilizable DO probe and the oxygensaturation was kept at 30% by regulating the stirring velocity between100 and 600 rpm. The aeration rate was set at IVVM.

Example 9 Full Thickness Evaluation

The metabolized cell extract disclosed above are tested on the MatTekfull thickness skin tissue model. This skin model consists of: 1) normalhuman-derived epidermal keratinocytes that have been cultured to form amultilayered, highly differentiated model of the human epidermis and, 2)human fibroblasts that have been seeded into a collagen matrix to formthe dermis. Upon arrival, the tissues are stored at 4° C. until used.For use, the tissues are removed from the agarose-shipping tray andplaced into a 6-well plate containing 4 ml of assay medium and allowedto equilibrate overnight at 37±2° C. and 5±1% CO₂. After the overnightequilibration, the media is replaced with 4 ml of fresh media and 50 μlof test material (i.e. metabolized cell extract) is then appliedtopically to the tissues. The tissues are then incubated for 24 hours at37+2° C. and 5+1% CO₂ and physiological effects of the treatmentmeasured over time.

Example 10 Preparation of Liposomal Encapsulated Metabolized ConditionedGrowth Medium

Samples of metabolized conditioned growth medium are incorporated intoliposome comprising phospholipid and lecithin layer obtained fromsoybeans. The lysate is slurried together with liposome using ahigh-pressure homogenizer obtained from Hydraulic EngineeringCorporation (Brea, Calif.). The milky white mixture contains themetabolized conditioned growth medium encapsulated with the liposomalcomponents.

Example 11 Preparation of Maltodextrin-Encapsulated Metabolized CellExtract

Samples of metabolized cell extract are encapsulated in maltodextrin andspray-dried to essentially provide an anhydrous powder ofmaltodextrin-encapsulated metabolized cell extract using themethodologies in WO2003/068161.

Example 12 Water-in-Oil Emulsion

The example illustrates a high internal phase water-in-oil emulsionincorporating the metabolized conditioned growth medium as disclosedabove.

Ingredient Wt % 1,3-dimethyl-2-imdazolidione 0.2 Polyoxylene (2) oleylether1 (Oleth-2) 5.0 Disteardimonium Hectorite 0.5 MgSO₄-7H₂O 0.3Preservative 0.01 Metabolized conditioned growth medium 10.0 Water To100

Example 13 Water-in-Oil Cream

The example illustrates a water-in-oil cream incorporating themetabolized conditioned growth medium prepared as disclosed above.

Ingredient Wt % Mineral oil 4 1,3-dimethyl-2-imdazolidione 1 Ceteth-10 4Cetyl alcohol 4 Triethanolamine 0.75 Butylene glycol 3 Xanthum gum 0.3Methyl, propyl and butyl paraben 0.01 Metabolized conditioned growthmedium 10 Water To 100

Example 14 Alcoholic Lotion

The example illustrates an alcoholic lotion incorporating themetabolized cell extract prepared as disclosed above.

Ingredient Wt % 1,3-dimethyl-2-imdazolidione 0.3 Ethyl alcohol 40.0Metabolized cell extract 10.0 Water To 100

Example 15 Sub-Micron Emulsion Concentrate

The example illustrates a sub micron emulsion concentration thatcontains the metabolized conditioned growth medium prepared as disclosedabove.

Ingredient Wt % Trimethylopropane Tricaprylate/Tricaprate 18.0 Glycerin8.0 Cetcaryl alcohol 2.0 Cetcareth 20 2.0 Glyceral stearate 2.0Butylated hydroxytoluene 0.01 Metabolized conditioned growth medium 10Water To 100

Example 16 Dilution of Metabolized Cell Extract

The example illustrates a dilution that contains the metabolized cellextract prepared as disclosed above

Ingredient Wt % Water 89 Metabolized cell extract 10 Preservative 1.0

Example 17 Human DNA Micro Array Studies on Normal Human DermalFibroblasts

Normal human dermal fibroblasts were treated for 24-hour exposure asfollows: (i) control 1: Nouricel (10×), i.e., conditioned growth medium(see, e.g., Example 2); (ii) control 2: Pichia ferment filtrate (20×),i.e., yeast ferment extract (see Example 8); (iii) partially fermentedNouricel (20×), i.e., partially metabolized conditioned growth medium(see Example 3); and (iv) fully fermented Nouricel (20×), i.e., fullymetabolized conditioned growth medium (see Example 3).

The results are presented in the table below and are represented as aratio of median between treated and untreated samples. A ratio ofmedian >1.3 indicates strong up-regulation and <indicates strongdown-regulation of the gene.

(ii) Pichia (iii) Partially (iv) Fully Ferment fermented fermentedSample (i) Nouricel Filtrate Nouricel Nouricel Gene Name COL1A1 1.531.114 1.013 1.12 COL1A2 1.608 1.994 1.371 1.806 COL4A1 0.978 0.443 1.1481.327 ELN 1.075 0.581 0.86 0.488 FN1 1.248 1.573 0.706 1.35 IVL 0.6750.316 0.256 0.5 PLOD1 0.981 1.196 0.855 1.417 TIMP1 1.874 1.852 1.3971.967 SOD1 1.184 1.396 1.215 0.965 SOD2 1.293 2.373 2.543 2.739 CAT0.902 0.294 0.678 0.48

Results from a second assay are presented in the table below and are asa ratio of median between treated and untreated samples. A ratio ofmedian >1.3 indicates strong up-regulation and <indicates strongdown-regulation of the gene.

(iv) Fully fermented Sample (i) Nouricel Nouricel Gene Name COL1A1 2.9141.388 COL1A2 3.2 2.102 COL4A1 0.957 0.714 VEGFB 1.223 1.339 TGF-Betal1.795 1.615 FGF3 2.284 1.867 PDGFRB 2.194 1.773 TJP1 2.302 1.804HSP90AB1 1.965 1.921 HSP90A1 2.871 2.05 HSPB1 1.947 1.881 ELN 1.5540.526 FN1 2.667 1.903 PLOD1 0.709 0.463 TIMP1 2.832 1.547 SOD1 1.3131.613 MMP3 0.601 0.660 MMP12 0.364 0.428 SOD2 1.293 2.594 CAT 0.6960.291

In one embodiment, the administration of the metabolized conditionedgrowth medium or the metabolized cell extract to a subject results inthe up-regulation of at least one extracellular matrix protein. In someembodiments, the extracellular matrix protein is up-regulated by about5% to about 100%, about 10% to about 90%, about 20% to about 80%, about30% to about 70%, about 40% to about 60%, or about 25% to about 50%. Inother embodiments, the extracellular matrix protein is up-regulated byabout 10% to about 50% or about 60% to about 100%. In yet anotherembodiment, the extracellular matrix protein is up-regulated by about5%, by about 10%, by about 15%, by about 20%, by about 25%, by about30%, by about 35%, by about 40%, by about 45%, by about 50%, by about55%, by about 60%, by about 65%, by about 70%, by about 75%, by about80%, by about 85%, by about 90%, by about 95% or by about 100%. In someembodiments, the extracellular matrix protein that is up-regulated is acollagen or lysyl hydroxylase protein. In some embodiments, theextracellular matrix protein is encoded by COL4A1 or PLOD1.

In one embodiment, the administration of the metabolized conditionedgrowth medium or the metabolized cell extract to a subject results inthe up-regulation of at least one repair protein. In some embodiments,the repair protein is up-regulated by about 5% to about 100%, about 10%to about 90%, about 20% to about 80%, about 30% to about 70%, about 40%to about 60%, or about 25% to about 50%. In other embodiments, therepair protein is up-regulated by about 10% to about 70%. In someembodiments, the repair-protein is up-regulated by about 5%, by about10%, by about 15%, by about 20%, by about 25%, by about 30%, by about35%, by about 40%, by about 45%, by about 50%, by about 55%, by about60%, by about 65%, by about 70%, by about 75%, by about 80%, by about85%, by about 90%, by about 95% or by about 100%. In some embodiments,the repair protein is fibronectin. In some embodiments, the repairprotein is encoded by FN1.

In one embodiment, the administration of the metabolized conditionedgrowth medium or the metabolized cell extract to a subject results inthe up-regulation of at least one cellular connectivity protein. In oneembodiment, the cellular connectivity protein is up-regulated by about5% to about 200%. In one embodiment, the cellular connectivity proteinis up-regulated by about 10% to about 80%. In some embodiments, thecellular connectivity protein is up-regulated by about 30% to about 50%.In one embodiment, the cellular connectivity protein is up-regulated byabout 5%, by about 10%, by about 15%, by about 20%, by about 25%, byabout 30%, by about 35%, by about 40%, by about 45%, by about 50%, byabout 55%, by about 60%, by about 65%, by about 70%, by about 75%, byabout 80%, by about 85%, by about 90%, by about 95%, by about 100%, byabout 110%, by about 120%, by about 130%, by about 140%, by about 150%,by about 160%, by about 170%, by about 180%, by about 190% or by about200%. In other embodiments, the cellular connectivity protein isinvolucrin protein. In some embodiments, the cellular connectivityprotein is encoded by IVL gene.

In one embodiment, the administration of the metabolized conditionedgrowth medium or the metabolized cell extract to a subject results inthe up-regulation of at least one antioxidant protein. In someembodiments, the antioxidant protein is up-regulated by about 5% toabout 300%, about 5% to about 200%, about 5% to about 150%, about 5% toabout 100%, about 10% to about 90%, about 20% to about 80%, about 30% toabout 70%, about 40% to about 60%, or about 25% to about 50%. In otherembodiments, the antioxidant protein is up-regulated by about 10% toabout 50% or about 200% to about 250%. In some embodiments, theantioxidant protein is up-regulated by about 5%, by about 10%, by about15%, by about 20%, by about 25%, by about 30%, by about 35%, by about40%, by about 45%, by about 50%, by about 55%, by about 60%, by about65%, by about 70%, by about 75%, by about 80%, by about 85%, by about90%, by about 95%, by about 100%, by about 125%, by about 150%, by about175%, by about 200%, by about 250% or by about 300%. In someembodiments, the antioxidant protein that is up-regulated is superoxidedismutase. In some embodiments, the antioxidant protein that isup-regulated is encoded by SOD2.

Example 18 In Vitro and Clinical Studies Patients and Methods Methods:In Vitro Study Gene Array Analysis

Human dermal fibroblasts were seeded into either a 96-well plate(cytotoxicity assay) or into T-25 flasks (cytokine array) and grown at37±2° C. and 5±1% CO₂ using fibroblast growth media (FGM). Safe workingconcentration for each test material was determined using an MTT assay.Upon reaching confluence, the cells were treated with the test materialsfor 24 hours. After the 24 hour treatment, total RNA was isolated usingan RNAqueous® Kit (Ambion) per the manufacturer's instructions. Afterpurification, the total RNA was prepared for array use by firstamplifying the RNA using a MessageAmp™ aRNA Kit (Ambion), and thenfluorescently labeling the aRNA with Cy3 or Cy5 using an ASAP LabelingKit (Perkin Elmer), both per the manufacturer's instructions. Thefluorescently labeled aRNA was applied to the DNA microarray chips(Agilent Technologies) and the chips were hybridized overnight andwashed per the manufacturer's recommended protocol. After washing, themicroarrays were scanned with an Axon GenePix 4100A Scanner and analyzedwith GenePix Pro software. Fluorescence intensities for the microarrayswere subjected to global normalization. The total fluorescent signal forboth dyes was normalized with a correction factor that would make theratio of total intensities for both dyes equal to one. For this study aCy3/Cy5 (untreated/treated) fluorescence intensity ratio greater than1.3 or less than 0.7 (this relates to a change in gene expression of atleast 30%) was used as the cutoff for up- and down-regulated genes,respectively. In addition, the fluorescence intensity of the gene markerhad to be greater than the background intensity.

Protein Array Analysis

Human dermal fibroblasts were seeded into either a 96-well plate(cytotoxicity assay) or into T-25 flasks (cytokine array) and grown at37±2° C. and 5±1% CO2 using fibroblast growth media (FGM). Safe workingconcentration for each test material was determined using an MTT assay.Upon reaching confluence the cells were treated with the test materialsfor 24 hours, after which cytokine release into the culture media wasassessed using cytokine arrays (Ray Biotechnology). The microarrays werescanned with an Axon GenePix 4100A Scanner analyzed with GenePix Prosoftware.

Methods: 12-Week, Clinical-Usage Study (Combination Product)

A 12-week open-label clinical study examined the efficacy andtolerability of the combination treatment serum in subjects with mild tosevere facial photodamage. The criteria for study participation includedfemale subjects aged 30 to 60 years (having a Fitzpatrick Skin Phototypeof I-IV) with clinically-determined mild to severe, fine and coarsewrinkles in the periocular area. Subjects were excluded from the studyif they had used topical retinoids within 3 months of the study start,received injections of dermal fillers or botulinum toxin or had facialpeels or had facial resurfacing procedures within 6 months or used otherforms of anti-aging products on the face within 30 days of study start.Subjects with known allergies to the facial product regimen were alsoexcluded.

Subjects were instructed to apply the treatment serum on their entirefacial skin, twice daily (morning and evening) for twelve weeks. Inaddition to the treatment serum, subjects agreed to the use of a basicskincare regimen, including a cleanser, light moisturizer and sunscreen.Subjects also agreed not to begin the usage of any new facial productsother than the provided materials for the duration of the study.

IRB approval was obtained for this open-label, single-center study fromIntegReview of Austin, Tex. The study was conducted according to ethicaland regulatory principals from the International Conference onHarmonization and good clinical practices.

Clinical evaluations were conducted at baseline (Visit 1), week 4 (Visit2), week 8 (Visit 3) and week 12 (Visit 4). The following procedureswere conducted at each visit.

Efficacy Assessments:

Fine and coarse wrinkles were clinically graded using a ten point scale,on each subject's right and left periocular area (where 0=none,0.5-3.5=mild, 4-6.5=moderate and 7-9=severe). Ten point scales were alsoused to assess skin tactile roughness (where 0=smooth, 9=rough), skintone (0=blotchy/uneven, 9=clear/even), skin firmness (where 0=skinappears loose and sags, 9=skin appears firm) and skin radiance(0=dull/flat matte, 9=bright/luminous/radiant). All grading assessmentswere performed by the same investigator at each visit to ensure gradingconsistency.

Safety Assessments:

Tolerability of the treatment product was assessed at all visits by thereporting of adverse events and by objective and subjective assessments.Objective irritation (erythema, edema and scaling) was assessed by theinvestigator whereas subjective irritation (burning/stinging, itchingand tingling) was assessed by the subject, all using a 4-point scale(where 0=none, 1=mild, 2=moderate and 3=severe).

Standardized Photography:

Standardized, digital images were taken of the subject's left, right andfrontal facial views using raking light (standard color).

Self-Assessment Questionnaires:

Subjects completed a Self-Assessment Questionnaire at Week 12, wherethey rated their facial skin condition and the treatment serum'sefficacy on a four point scale (Strongly Agree, Agree, Disagree,Strongly Disagree). Subjects rated their overall satisfaction with thetreatment serum on a four point scale where 1=Excellent, 2=Good, 3=Fairand 4=Poor.

Statistical Analysis:

Mean clinical grading scores at weeks 4, 8 and 12 were compared to meanbaseline scores using a student's paired t-test. Changes from baselinewere considered significant at the p<0.05 level. For the subjectSelf-Assessment Questionnaires, the percent incidence of positiveresponders was reported for the treatment serum's efficacy and aestheticattributes sections.

Results Results: In Vitro Study

After treatment of human dermal fibroblasts with Biometa Complex (yeastmetabolized conditioned growth media), the gene expression profiled wasanalyzed with a special emphasis on genes implicated in the anti-agingcascade. The following Table lists some of the key genes regulated byBiometa complex: Biometa complex stimulates a number of genes involvedin the extracellular matrix regeneration.

Function Genes Brief Description Extracellular Col 1A1, COL12A1, Genesencoding collagen Matrix Proteins COL15A1, Growth Factors TGF-BetaInduces synthesis and secretion of and Cytokines major extracellularmatrix proteins, involved in collagen and elastin repair and ECMTIMP1/TIMP3 TIMPs play an important role in the production regulation ofMMP activity VEGF-A/VEGF-B Key role in angiogenesis FGF3 Enhancedhyaluronan production and implications in tissue remod- eling PDGFRBImplicated in tissue remodeling IGF Enhanced hyaluronan production andimplications in tissue remod- eling IL-10RB, IL-11, Involved in severalsignal transduc- IL-8, etc. tion pathways that include tissueremodeling, etc Antioxidant SOD1 involved in regulation of ROS- mediatedtissue damage; usually found in the extracellular matrix and is ideallysituated to prevent cell and tissue damage initiated by reactive oxygenspecies (ROS) Stress Related HSP Heat shock proteins are part ofcellular defense against stress Skin Barrier TJP1/ZO-1 Help improve skinbarrier function

Additionally we saw a down-regulation of MMP (enzyme responsible forbreaking down collagen and elastin). This down-regulation of MMPcorrelated well with an upregulation of TIMPS (a natural inhibitor ofMMPs). Biometa complex has also upregulated other genes necessary tomaintain the overall health of the skin including stress proteins (HSP),antioxidant proteins (SOD1), and proteins important for maintaining skinbarrier (TJ1).

Biometa Complex also stimulated fibroblasts to express and synthesizekey proteins (cytokines, growth factors, etc.) known to be involved inskin repair and remodeling including bFGF (Fibroblast Growth Factor),TIMP-1 (Tissue inhibitor of Metalloproteinases), and VEGF (Vascularendothelial growth factor) based on analysis of proteins released intomedium after treatment. This data shows a good correlation between geneexpression and protein synthesis. These results demonstrate that Biometacomplex has the ability to stimulate fibroblasts, thereby reducing signsof aging.

Results: 12 Week Clinical-Usage Study

Thirty-three female subjects aged 35-56 years with mild to severe, fineand coarse periocular wrinkles were enrolled and completed the twelveweek study.

At weeks 4, 8 and 12, statistically significant improvements in meanscores for all photoaging parameters were achieved after twice-dailytopical use of the combination treatment serum. Investigator assessmentsdemonstrated significant reductions in mean scores for fine and coarseperiocular wrinkles at week 4 with continued significant reductionsthrough week 12 (all P<0.0001) as shown in FIG. 4. Most notably, meanscores for coarse periocular wrinkles decreased by 30% at week 12. Inaddition, mean scores for skin firmness, skin tone, radiance and tactileroughness reflected similar significant and progressive improvementsfrom baseline and at all subsequent follow-up visits (all P≦0.02) asdepicted in FIG. 5 and FIG. 6. Especially prominent was the improvementin the texture of the skin achieved as early as week 4, shown by asignificant reduction (79.5%) in mean tactile roughness scores(P<0.0001).

The subject responses in the Self-Assessment Questionnaire at week 12strongly support the improvements observed in the investigatorassessments, with over 85% of subjects responding favorably to allquestions. The percentage of subjects who selected Strongly Agree orAgree in response to the questionnaire at Week 12 are presented in FIG.7.

Standardized digital photographs (data not shown) represent examples ofclinical responses after twice-daily use of the combination treatmentserum. Briefly, improvements in peri-ocular fine and coarse wrinkles andskin tone were demonstrated in a 49 year old Caucasian female subjectafter only four weeks of product use. In addition, a 46 year oldHispanic female subject showed visible improvements in fine peri-ocularwrinkles, skin tone, tactile roughness and radiance at week 4. A 46 yearold Korean/Caucasian female subject presenting with fine and coarseperi-ocular wrinkles and uneven skin tone visibly improved after fourweeks of product use.

The combination treatment serum was well-tolerated and there were notreatment-related adverse events reported during the course of thestudy. Mean scores for edema, scaling, burning/stinging, itching andtingling remained less than mild throughout the study (all scores <0.09on the 0-4 scale). Notably, mean scores for erythema decreased frombaseline at all follow-up visits, with a statistically significantdecrease observed at week 8 (P<0.03).

CONCLUSION

This treatment serum provides physicians and patients with a unique andeffective topical combination product that improves signs of skin aging.

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications in additionto those described will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A process for preparing a metabolized conditionedgrowth medium, the process comprising: (a) culturing cells in a growthmedium sufficient to meet the nutritional needs required to grow thecells in vitro to form a conditioned growth medium and removing theconditioned growth medium from the cultured cells; (b) culturing yeastcells; (c) exposing the yeast cells to the conditioned growth medium;(d) culturing the yeast cells to metabolize at least a portion of theconditioned growth medium, and; (e) collecting the metabolizedconditioned growth medium.
 2. The process of claim 1, wherein thecollected metabolized conditioned growth medium of step (e) isconcentrated, filtered and/or purified.